Open Access Scientific Reports

Gastrointestinal Tract Abnormalities in Autism, Inflammatory Bowel Disease and Many Other Clinical Entities May Be Due To T. Gondii infection

Research Article Open Access
Joseph Prandota*
Department of Social Pediatrics, Faculty of Health Sciences, University School of Medicine, Wroclaw, Poland
*Corresponding authors: Joseph Prandota Department of Social Pediatrics Faculty of Health Sciences University School of Medicine Wroclaw, Poland E-mail: jozef.prandota@am.wroc.pl
Received March 16, 2012; Published August 12, 2012
Citation: Prandota J (2012) Gastrointestinal Tract Abnormalities in Autism, Inflammatory Bowel Disease and Many Other Clinical Entities May Be Due To T. gondii infection. 1: 256. doi:10.4172/scientificreports.256
Copyright: © 2012 Prandota J. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Several studies demonstrated various gastrointestinal tract (GT) disturbances in children with autism, including active/chronic inflammation of the GT and development of ileo-colonic lymphoid nodular hyperplasia. Emerging evidence now indicates that autism and many congenital and acquired GT abnormalities and their intensity, including Hirschsprung’s disease, may be due to prenatal and/or postnatal damage of the enteric nervous system associated mainly with peroral infection with T. gondii, its genotype, virulence/antigenicity, number of oocysts/sporozoites/ bradyzoites/ tachyzoites, part of GT tract infected, and the host innate prenatal and postnatal immunity. Maternal and fetal microchimerisms and physiologic swallowing of amniotic fluid by the fetus probably play an important role in early dissemination of the parasite as a Trojan horse and development of these abnormalities. Mast cells are vital in sustaining gut inflammation because they are infected with the parasite, secrete chemotactic factors able to recruite neutrophils, macrophages and lymphocytes when the parasite reaches the lamina propria, and finally reduce survival and cause death of myenteric neurons. Lactoferrin, a component of the breast milk, plays an important role in the host defense against T. gondii infection and dissemination, therefore it should be included into therapeutic assets used for treatment of GI tract disturbances caused by the parasite both in young and adult patients.
Keywords
T. gondii infection; Autism spectrum disorders; Gastrointestinal tract pathology; Ileocolonic nodular hyperplasia; Enteric nervous system; Myenteric neurons; Inflammatory bowel disease; Necrotizing enterocolitis; Lactoferrin.
Gastrointestinal Disturbances in Patients with Autism Spectrum Disorders (ASD)
Autistic children frequently develop several gastrointestinal (GI) symptoms including pyrosis (heart burn), regurgitations, excessive salivation, vomiting, constipation, fecal impaction, chronic diarrhea, abdominal discomfort and pain, gaseousness, and distention [1-11]. Recent review by Buie et al. [12] based on 11 studies reported that the prevalence of GI symptoms, such as constipation, diarrhea, bloating, belching, abdominal pain, reflux, vomiting and flatulence, in children with ASD range widely from 9 to 91%, averaging 44%.
Horvath et al. [8] found reflux esophagitis in 60% of an autistic sample, duodenal inflammation in 67%, low carbohydrate digesting enzymes (lactase) in 58%, and abnormal pancreatic response to secretin in 75% of the individuals. Gonzales and his group [10,11] reported that 100% of their 45 ASD children had chronic inflammation and lymphoid nodular hyperplasia (LNH) in the colon compared with 66.66% of the 57 developmentally normal controls, reflecting a high background rate of infectious enterocolitis in Venezuelan young patients. Gastrointestinal endoscopy showed that these children had also reflux esophagitis (88.88% vs. 48.71%, P < 0.001) and eosinophilic esophagitis, nonspecific gastritis (55%) with micro- and macronodularity in the gastric body and antrum, nonspecific inflammation of the small intestine (37%), including chronic active duodenitis with LNH, and intestinal villus alterations [10,11].
Ingested foreign antigens enter the gut mucosa through the microfold (M) cells in the Peyer’s patches or through damaged epithelium, and are taken up by antigen presenting cells, most likely dendritic cells (DCs). DCs move to Peyer’s patches and mesenteric lymph nodes, where they interact with naive lymphocytes and initiate adaptive immune response that result in activation of T and B memory cells, and proliferative response and cytokine release, finally leading to GI inflammation [13,14]. Enhanced density of dendritic T cells was observed in the colon of ASD children with GI tract disturbances compared to controls [6]. (Nb. it should be noted that the hostendoplasmic reticulum-parasitophorus vacuole interaction provides a route of entry for antigen cross-presentation in T. gondii-infected DCs [15]). Chronic inflammation in the gut can damage the epithelial cell layer, and D’Eufemia et al. [5] showed abnormally increased intestinal permeability in 9 out of 21 analyzed autistics (43%) but in none of the 40 controls [8]. de Magistris et al. [16] also reported abnormally high intestinal permeability in their sample of patients with ASD and their first-degree relatives compared with controls (36.7% and 21.2% vs. 4.8%, respectively).
In several autistic patients Ashwood and Wakefield [9] reported increased levels of proinflammatory cytokines TNF-α and IFN-γ, and reduced levels of regulatory IL-10 cytokine in peripheral blood and mucosal lymphocytes. PBMCs from ASD children stimulated with LPS produced higher levels of TNF-α than controls regardless of dietary interventions [17]. In PBMCs from ASD children with positive GI symptoms, Jyonouchi et al. [17] found a positive association between TNF-α levels produced by LPS and those with cow’s milk protein. In the unrestricted diet group, GI positive ASD PBMCs produced higher IL-12 than controls and less IL-10 than GI negative ASD PBMCs with LPS. Furthermore, some studies suggested an association between gut inflammation and NO-dependent oxidative injury [18]. Recently, it was shown that NO metabolite S-nitrosogluthatione, a novel potent inducer of intestinal barrier function in human colon [19] is secreted by enteric glia cells [20]. It is possible that the increased NO levels in autism may also be responsible for the gastrointestinal abnormalities observed in some individuals with autism [21]. This is in line with the finding that patients with autism have increased NO levels and glutathione peroxidase activity in red blood cells (P < 0.0001) [22], and the incubation of sodium nitroprusside (SNP) with Caco-2BBe intestinal epithelial monolayers resulted in time-, and concentration-dependent decreases in transepithelial resistance [23]. Electron microscopy study revealed that SNP widened tight junctions. NO reduced cellular ATP levels and reversibly increased permeability of tight junctions in cultured Caco-2Bbe cells [23]. Moreover, Unno et al. [24] demonstrated that incubation of cultured human intestinal epithelial monolayers Caco- 2BBe cells with IFN-γ resulted in upregulation of NO biosynthesis and a marked increase in permeability of intestinal epithelial monolayers.
Wakefield et al. [3] reported prominent ileal LNH and ileocolitis called “autistic enterocolitis”, in an uncontrolled study of 12 autistic children. Mucosal lesions manifested as chronic ileocolonic LNH were characterized by lymphocyte infiltration, complement deposition, and cytokine production. This inflammatory condition was associated with eosinophilic infiltrate lesions on the intestinal wall, in the upper gastrointestinal tract [18]. It was found [25] that the prevalence of LNH was significantly greater in ASD children compared with controls in the ileum (90 vs 30%, P < 0.0001) and colon (59 vs 23%, P = 0003). The severity of ileal LNH was markedly greater in ASD children compared with controls, with moderate to severe ileal LNH present in ASD children (68 vs15%, P < 0.0001). Severe ileal LNH was associated with co-existent colonic LNH. The presence and severity of ileal LNH was not influenced by either diet or age at colonoscopy and hyperplastic lymphoid follicles were significantly more prevalent in the ileum of ASD children compared with controls (61 vs 9%, P < 0.0001). The authors suggested that ileo-colonic LNH was a characteristic pathological finding in children with ASD and gastrointestinal symptoms, associated with mucosal inflammation [25]. It is interesting that Kokkonen & Karttunen [26] analyzing a consecutive series of 140 children with persistent and severe gastrointestinal symptoms revealed that among the whole study group 46 subjects also had LNH, and 26% could be defined as having food allergy. This expression of mucosal immune response on the mucosa of the colon or terminal ileum, the authors considered as common but not an innocent bystander, which may be related to food allergy but also to other immunologically active disease states [26].
In ASD children, histopathologic examination showed lymphocytic colitis, although less severe than in classical inflammatory bowel disease (IBD) [3]. Basement membrane thickness and mucosa gamma cell density were significantly increased as compared with controls, inlcuding IBD. Intraepithelial lymphocyte numbers and CD3, plasma cell, and CD8 cell counts were also markedly increased [3]. The authors concluded that IBD was a lymphocytic enterocolitis in ASD, probably skewed in the TH2 dominant direction [3]. Torrente et al. [7] also analyzed duodenal biopsies in 25 children with regressive autism and found increased number of enterocytes and Paneth cells, duodenal lining demonstrated increased lymphocytic proliferation, crypt cell proliferations, and more T cells. Also, the increased IgG deposition on the epithelial cell surfaces accompanied by complement C1q have been found [7]. The authors believed that this pattern was characteristic for autism. The serum levels of complement C3/C4 proteins and α1- antichymotrypsin (both these biomarkers are positive acute phase proteins in blood that facilitate immunological and inflammatory responses) were found to be markedly higher in children with autism than in their unaffected siblings [21,27,28]. Duodenal biopsies performed by Kushak et al. [29] showed that frequency of lactase deficiency was 58% in autistic children ≤ 5 yrs old and 65% in older patients. However, only 6% of autistic patients had intestinal inflammation, and it was suggested that lactase deficiency may contribute to abdominal discomfort, pain and observed abnormalities in behavior of those children [29].
The presence of LNH has been found also in normally developing children with autism and gastrointestinal symptoms [6]. Histologic studies demonstrated lymphocytic colitis less severe than in IBD. Basement membrane thickness and mucosal gamma delta cell density were significantly increased above those of subjects with Crohn’s disease, IBD, ulcerative colitis (UC), and children with LNH. CD8+ density and intraepithelial lymphocyte numbers were higher than those in the Crohn’s disease, LNH, and normal control groups. CD3 and plasma cell density and crypt proliferation were higher than those in normal and LNH control groups. Epithelial glycosaminoglycans were disrupted, but the epithelium was HLA-DR (-), suggesting a predominantly TH2 response [30]. It is interesting that glucosamine, a naturally-occurring amino monosaccharide, has been shown to exhibit an antiinflammatory action by inhibiting neutrophil functions. Moreover, this monosaccharide suppressed IL-8 production and ICAM- 1 expression by TNF-α-activated human colonic epithelial cell line HT- 29 cells [30], as well as it inhibited TNF-α-induced phosphorylation of p38MAPK and NF-κB p65, and nuclear translocation of NF-B in intestinal epithelial cells, which may confirm its antiinflammatory activity [30]. Wang et al. [31] found that IL-6 receptors are present in intestine epithelia in a polarized fashion, and basolateral IL-6 and, to a lesser extent, apical IL-6 induced activation of the NF-κB pathway. IL-6 induced polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. ICAM- 1 induction by IL-6 required activation of NF-κB. They demonstrated that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulated IL-6-induced NF- κB activation and ICAM-1 expression [31]. These are important informations because patients with ASD have systemic manifestions of the immune deregulation/chronic inflammatory condition with elevated levels of several proinflammatory cytokines, including IFN-γ [32], TNF-α [33], IL-6, IL-8, IL-12 [34], NO and other biomarkers (Table 1). An ongoing relevant inflammatory response in children with autism has been suggested by the elevated macrophage product neopterin found in these individuals [48]. Otherwise, it must be noted that astrocytes secretion of IL-1 and IL-6 upon infection was triggered by T. gondii bradyzoites and tachyzoites in a time-, and dose-dependent manner [49,50]. In addition, IL-6 may enhance T. gondii intracellular multiplication also in a dose-dependent manner [51,52] therefore these activities may drive local and systemic inflammatory reactions. Moreover, inhibited production of antiinflammatory cytokines, such as IL-10 [42] and TGF-β [43] found in children with autism suggested also a deficiency of natural feedback inhibitor mechanisms, albeit it must be emphasized that TGF-β is a potent deactivator of PMN and macrophages since it suppresses the production of ROS, RNI and IL-1, as well as impair expression of L-selectin on PMN known to be essential for PMN recruitment [53], and increases T. gondii replication also significantly enhancing TGF-β production [54].
Finally, Medical Research Council [55] suggested that LNH could also be a secondary phenomenon, related to infections or infestations. The histological appearances of the ileal biopsies commonly included reactive follicular hyperplasia, marked expansion of lymphoid tissue, and acute cryptitis; ileitis, eosinophil infiltration and an increase in intraepithelial lymphocytes (IELs) were unusual [56]. In the colon, biopsies showed appearances that were similar to, but less severe than those seen in children with established ulcerative colitis, being perhaps more reminiscent of the features of lymphocytic colitis, as seen in adults [57]. It is of interest, given the proposed association of autism with gluten intolerance, that colonic inflammation has been also described in adults with celiac disease [56,58].
Morphometric Abnormalities of the Enteric Nervous System (ENS) in Experimental Animals Caused by T. gondii Infection may be also At Least in Part Responsible for Development of Gastrointestinal Tract Dysfunction Reported in Patients with Autism and Individuals with IBD
Development of the ENS
The ENS is an independently acting nerve network within the walls of the gastrointestinal tract that controls secretion, motility, blood flow, uptake of nutrients, immunological and inflammatory processes in the gut [59]. Two main cell populations are represented in the ENS, enteric glial cells (EGCs) and neurons, the former being up to 4-fold more abundant than neurons [60,61]. Glial cells are found encapsulating neuronal cell bodies within the enteric ganglia and also surrounding the neurons that project from this plexi to innervate the intestinal mucosa and submucosa [62]. In humans, the ENS is subdivided into several plexuses (subserous, longitudinal muscle, myenteric, circular muscle, deep muscle, muscularis mucosae, and mucosal) [63]. Ganglionated plexuses are present in the submucosa (Meissner’s and Henle’s plexuses) and in the septum between the circular and longitudinal layers of the muscularis propria (Auerbach’s plexus) [64]. EGCs are small cells with a “star-like” appearance containing intracellular arrays of 10 nm filaments made up of glial fibrillary acid protein (GFAP) [65,66]. Most EGCs are found within the ganglia, and are also present in the interconnecting nerve strands of the ganglionated and in all nonganglionated plexuses [67,68]. In the ganglia, EGCs are very tightly packed around neurons [69,70]. The ENS has many interneurons and intrinsic microcircuits [71]. The ultrastructure of the ENS is different from those of sympathetic or parasympathetic ganglia, enteric neurons are supported by glia rather than by Schwann cells, enteric ganglia lack internal collagen, and the ENS resembles the CNS more than it resembles other regions of the peripheral nervous system [72-74].
The ENS comprises the neurones and glia that are found in the wall of the gastrointestinal tract [75-79]. Its ganglia contain primary afferent neurones, interneurones and motor neurones, i.e. the neural components necessary for complex reflex circuitry [71,75,76]. The gastrointestinal tract is also home to the largest component of the immune system in the body, which serves to defend the host from viral, bacterial, or parasitic invasion, and to limit the consequences of the antigenic stimulus caused by the digestion of food. The submucosal and myenteric plexuses of the ENS contain a large number of neurones, but enteric neurones are outnumbered by enteric glia [79,80]. Glial cells in the ENS appear to be very similar in origin, gross morphology and ultrastructure to astrocytes of the CNS and bear similar relationships with neuronal cell bodies and processes to peripheral Schwann cells [60]. Enteric glia are connected to one another and to enteric neurones via gap junctions, and their processes ensheat enteric neurones and can project to blood vessels and the mucosa [60,81]. Enteric glial cells (but not neurones) contain L-arginine immunoreactivity, suggesting that that NO precursor is supplied to enteric neurones by glial cells [82]. In addition to enteric neurons, enteric glia express the GABA reuptake transporter GAT-2, which suggests the participation of enteric glia in the removal of released neurotransmitters from the synaptic cleft [83].
Neuronal loss associated with aging
Studies in rats showed a correlation between small intestine length with body weight [84]. On the other hand, Schäfer et al. [85] believed that the intestinal length and muscular layer thickness were related to the development of the intrinsic innervation, and the increase in these parameters would provoke changes in neuronal density. Recently, Marese et al. [86] suggested that both these observations are correct, emphasizing the importance of the relation of intestinal length to neuronal density with more advanced age (Table 2). Investigations in animal models showed striking neuroplasticity in the ENS associated with aging [86-88]. A linear decrease in neurons number with age in all small intestine segments, being the presented in duodenum up to 30% [86,88] (Table 3). A probable cause of this reduction was attributed to the reduction in the integrity of the extrinsic vagal innervation, which may make the intrinsic network more susceptible to the effects of the advance of age, and approximately 20% of the myenteric neurons of the duodenum have afferent vagal innervation [86]. Neurons number reduction of the ENS related to age were also observed in the gastrointestinal tract of humans [89,90]. Moreover, some neurons can be more susceptible than others [91], and this may be related to the increase in ROS production [92]. It appeared that positive NADPHdiaphorase neurons, which express NO activity (nitrergic neurons) are less vulnerable to the process of aging than cholinergic neurons [93-95]. Phillips et al. [94] showed that myenteric neurons in the large intestine of rats were significantly larger at 24 months and numerous NADPHdiaphorase positive axons were swollen. In younger animals (21 and 60 days old), Marese et al. [86] demonstrated a predominance of neurons with a smaller area (0-200 μm2), while neurons with a larger area of the cellular profile (200-300 μm2) predominated in animals of higher age groups. These changes probably were responsible for maintenance of ganglionic organization in all the age groups possibly because the larger neurons have a higher capacity of synthesis (reflected by Giemsa technique) and transport (Myosin-V) [86] (Table 4).
Table 1: Elevated biomarkers of neuroinflammation in autism (acc. to Rossignol [35]; with own modification).
Table 2: Body weight, length of small intestine and thickness of the external muscle tunica of the duodenum of rats of different ages (acc. to Marese et al. [86]; with own modification).
Table 3: Neuronal quantification by the non-histochemical Giemsa and the Myosin-V immunohistochemical techniques (n = 10) in an area of 14.832 mm2 of the duodenum of rats, and values converted into cm2 (acc. to Marese et al. [86]; with own modification).
Table 4: Area of the myenteric neuronal cell body profile (m2) estimated by Giemsa and Myosin-V techniques (n = 10) (acc. to Marese et al. [86]; with own modification)
Damage of enteric glia caused by peroral prenatal infection with T. gondii may be, at least in part, responsible for development of necrotizing enterocolitis in young infants
Hirschsprung’s disease is the most visible congenital birth defect of the ENS, in which ganglia are totally absent from variable lengths of the terminal bowel [96]. It must be noted that the ENS is responsible for the integrative control of behavior [97,98], and vagus nerve stimulation can be employed to affect epilepsy [99,100], treat depression [101,102], and even to improve learning and memory [103]. Serotonin may be a late acting growth factor promoting the development of subsets of enteric neurons and muscle/interstitial cells of Cajal (ICCs), and enteric serotonergic neurons are an example of neuron that might well cause a sublethal disorder of intestinal motility when they develop abnormally [104].
Schreiner et al. [105] suggested that it would be interested to investigate wheather oral infection with T. gondii is associated with IBD in humans. Until now, a slightly but significantly higher seropositivity rate based on Sabin-Feldman dye test results has already been observed in a small cohort of patients with Crohn’s disease over age 40 in Israel [106].
An important role for EGCs in gastrointestinal tract inflammation may be supported by the studies performed in two transgenic mouse models in which the ablation of enteric glia led to fulminant and ultimately fatal small and large intestinal inflammation that was unrelated to bacterial overgrowth [107,108]. One cannot therefore exclude that an exaggerated inflammatory response [109-112] mounted abnormally by the immature intestinal cells of the premature infants to gastrointestinal injury and caused by prenatal oral T. gondii infection sustained in the neonatal period resulted in development of necrotizing enterocolitis. Abnormal bacterial colonization, genetic predisposition [113-115], and various milk formulas [116] may be important contributing factors.
Glia in the CNS react to inflammatory insults with proliferation, increased cytokine secretion, and expression of GFAP, a phenomenon known as reactive gliosis [79]. The expression of GFAP has been reported to be higher in enteric cells compared with CNS glia [117]. In the CNS, proinflammatory cytokines, such as IL-1β and TNF-α have been shown to elicit astrocyte proliferation [118-120], whereas the anti-inflammatory cytokine IL-10 has been found to inhibit astroglial reactivity in vivo [121-123]. The proliferation rate of enteric glia also can be modulated by cytokines, for example IL-1β significantly and dose-dependently suppressed EGCs proliferation, while IL-10 had a biphasic effect, suppressing cell proliferation at lower concentrations and augmenting it at higher concentrations [124]. EGCs may act as “receptors” for cytokines and they themselves produce IL-6 and IL-1β [125,126], and express iNOS and L-arginine, the machinery for the time-delayed and micromolar release of NO [85,127]. Stimulation with TNF-α also involved in intestinal inflammation, did not affect glial IL-6 mRNA expression, while IL-1β stimulated IL-6 mRNA and protein synthesis in a time-, and concentration-dependent fashion [125].
It must be noted that children with ASD have evidence of neuroinflammation [36,37,128] and marked activation of microglia and astroglia with elevation of IL-6 and macrophage chemoattractant protein-1 (MCP-1) were found in autistic brain samples and cerebrospinal fluid [36]. In addition, some autistic children have increased GFAP in brain samples and CSF [37,38] (Table 1). It should be noted that studies of Matowicka-Karna et al. [129] performed in women with peripheral lymphadenopathy due to acute infection with T. gondii (IgM index > 0.7, specific anti-T. gondii IgG titer exceeded 300 IU/ml, low avidity) also showed highly significant increase of serum IL-5, IL-6, and IL-10, while TNF-α level was not changed. All these findings may at least in part suggest that there was a link between oral infection with T. gondii and the development of ileo-colonic LNH in children with ASD [25].
Abnormalities of the ENS reported in IBD
There is increasing evidence implicating enteric nerves in the pathogenesis of IBD [130]. Full-thickness biopsy of the jejunum revealed inflammation and myenteric ganglioneuritis in severe IBS (low-grade infiltration of lymphocytes in the myenteric plexus, which had peri- and intraganglionic location, intraepithelial lymphocytosis with cytotoxic T-cell predominance, hyperplasia and hypertrophy of Cajal cells, neuron degeneration, and the longitudinal muscle layer thickened) [131,133]. Structural abnormalities of the ENS and even outside the ENS have consistently been observed in CD and, less frequently, in UC [134]. Recently, Villanacci et al. [135] demonstrated a marked increase for neuronal cell bodies, enteroglia and ICCs in deep muscular plexus in CD while in uninvolved areas the number of enteroglial cells was decreased. In UC an increase of ICCs in the muscular propria and enteroglial cells was found in the diseased tissue. Myenteric plexitis was observed in 75% of CD (transmural disease), and 56% of patients with UC (mucosal disease) [143] even in macroscopically normal intestinal segments [136]. Ohlsson et al. [136] found enteric ganglioneuritis in 11 of 19 studied patients with CD and in 5 of 11 with UC, and only individuals with CD had ganglioneuritis in the small intestine. Moreover, in CD the ICCs in the small bowel showed atrophy and vacuolar degeneration, along with the significantly reduced number of cells (P = 0.005); in UC, the colonic ICCs were hyperplastic (P = 0.05) without signs of degeneration [136]. Ultrastructural abnormalities of ICCs in these clinical entities, such as swelling of mitochondria, decreased electron density autophagosomes and partial depletion of cytoplasm, were also described in previous studies [137,138]. Structural abnormalities of the ENS and neurochemical changes in IBD patients are presented in Tables 5 and 6.
Table 5: Structural abnormalities of the enteric nervous system in inflammatory bowel disease (acc. to Geboes et al. [134]; with own modifications).
Table 6: Neurochemical changes in inflammatory bowel disease (acc. to Vasina et al. [169]; with own modification).
Vasculitis, granulomatous arteritis and lymphangitis are not uncommon in CD [179,180]. Enteroglial cells surrounding the nerves innervating mucosal blood vessels show MHC class II expression during inflammation [166]. Disturbances of the cellular components of the ENS found in CD and UC included hyperplasia or an increase in the number of neuronal cell bodies, mainly in the ganglia of the submucosal plexuses, neuronal cell damage, and neuronal hypertrophy. Hypertrophy of neurons with the appearance of prominent organelle, including numerous strands of rough endoplasmic reticulum has been identified in tissue samples obtained from patients with CD [152,155]. Neuronal hypertrophy in Meissner’s plexus seemed to be more common in UC than CD, while hyperplasia of neuronal cell bodies was a more frequently observed and constant finding than neuronal hypertrophy [142,145,146,156]. A 3-fold increase in the number of ganglion cells of the ileal myenteric plexus was recorded in a series of 24 cases with CD. The increase was also present in areas not actively involved in the inflammatory process [157]. Increased numbers of ganglion cells were reported for the submucosal plexuses in CD when compared with control samples [158]. Neuronal hyperplasia of the myenteric plexus was also reported for UC, but the data sometimes may have been confused by difficulties in the differential diagnosis between granulomatous colitis and genuine UC [153,181].
In CD, a relative increase in NO positive neurones has been demonstrated in the submucosal and myenteric plexuses [159,165]. The increase in myenteric neurones containing NOS and also vasoactive intestinal peptide (VIP) and PACAP might cause persistent relaxation of smooth muscle of the affected segment(s) [159]. An increase in number, size and immunostain and an abnormal pattern of VIPcontaining fibers have been reported in CD [158,161,162]. The increase was found in histologically normal bowel, in areas with pathology both in specimens of the ileum and colon. In addition, an increase in the number of VIP immunostained ganglion cells in the submucosus plexus with 6-8 immunoreactive cell bodies in the ganglia in the CD samples and 0-2 in controls was noted. Numbers of immunoreactive neurones in the myenteric ganglia were 7-89 for VIP in CD compared to 2-45 in controls and 7-65 compared to 4-73 for NOS [159]. A marked decrease in V nerve fibers was found in the lamina propria and submucosa in both UC and CD, and in the lamina propria the variation in decrease was significantly associated with the severity of the disease [163,164].
Ultrastructural changes in the nerve elements in Crohn’s disease
Ganglion cell and axon degeneration and necrosis has been demonstrated in the small bowel autonomic nerve plexus of patients with Crohn’s disease [149,150], and it was suggested that this feature may be useful in differential diagnosis of Crohn’s disease from other conditions, especially ulcerative colitis [150]. Brewer et al. [151] also found considerable numbers of abnormal, very small axons, and concluded that axonal damage is common in chronic IBD, but is not specifically related to Crohn’s disease. In animal model, Bradley et al. [183] showed that inflammation did not stimulate appearance of new myenteric neurons of the guinea-pig ileum but stimulated mitosis in myenteric glia. Electron microscopic examinations showed a markedly decreased number of synapses in the wall of small intestine in Crohn’s disease [184]. In the ileum, the numbers of nerve terminals were decreased, as well as that of the vesicle population in the remaining nerve terminals. Some of the nerve processes were degenerating, and the number of the lysosomes in the nerve cell bodies increased. Inflammatory cells, such as lymphocytes, plasma cells, and mast cells were demonstrated in tola submucosa and in the mucous membrane, and their number was also increased. It was suggested that these immune cells and their bioproducts were responsible for these neuronal abnormalities in the small intestine in Crohn’s disease [184].
Enteric glial cells (EGCs) and intestinal inflammation
Animal studies showed that ECGs may play an important role in intestinal inflammatory processes [185-187], and that initiation and/ or progression of IBD (especially Crohn’s disease) might be ascribed to an immune-mediated damage of enteric glia [108]. The finding that EGCs functionally interact with lymphocytes [125], respond actively to inflammation process, and become activated as antigenpresenting cells [188] attracting immune cells to the ENS [60,185] suggests that this cell population is likely involved in inflammatory processes in the gut. Studies performed in patients with IBS showed the presence of inflammatory infiltrates closely associated with the enteric plexuses and mucosal activation of the immune system [131,189], and some individuals with intestinal dysmotility and megacolon have a lymphoplasmacellular infiltrate within the myenteric plexus that likely accounts for their symptoms [132].
Development of the Gastrointestinal Tract Immunity in Newborns and Young Children
Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. There are qualitative and quantitative age-specific changes in innate immune reaction in response to various stimuli. Neonatal B cells do not respond to polysaccharide antigens to produce antibodies required for neutralization, opsonization, phagocytosis and complement activation (Tables 7 and 8). This may explain why young children are particularly vulnerable to infections caused by encapsulated bacteria. However, the conjugation of polysaccharides to protein carriers allows T-cell recruitment and renders the polysaccharides immunogenic in early infancy [195,196].
Table 7: Age related functional characteristics of systemic B cell (antibody) response in early childhooda (acc. to Ogra [190]; with own modification).
Table 8: Age-related development of cellular characteristics of human Peyer’s patches (acc. to Ogra [190].
It was reported that neonatal innate toll-like receptor (TLR)- mediated responses are distinct from those of adults [197]. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells, produced less IL- 12p70 and IFN-α (and consequently induced less IFN-γ), moderately less TNF-α, but as much or even more IL-1β, IL-6, IL-23, and IL-10 than adult cells. Corbett et al. [198] summarized the innate TLR responses of human blood mononuclear cells and found that:
1. TH17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood;
2. antiviral defense (IFN-α) reached adult level function by 1 year of age;
3. TH1 type immunity (IL-12p70, IFN-γ) slowly rose but remained far below adult responses at 2 years of age;
4. IL-10 production steadily declined from from a high around birth to adult levels by 1 or 2 years of age; and
5. production of TNF-α or IL-1β varied by stimuli [198].
These changes support TH17- and TH2-type immunity promote defense against extracellular pathogens, and have a reduced capacity to reinforce TH1-type responses, which promote defense against intracellular pathogens [197]. In this context, it must be noted that TLR9 expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, is required for the gut-associated effective development of lymphoid tissue TH1-type immune response following oral infection with T. gondii [199]. Murine mesenteric lymph node dendritic cells also showed protective mucosal TH2 immune response against the parasite, including a production of specific secretory IgA [200]. In addition, innate recognition by TLR4 was involved in protective mechanisms against peroral infection with T. gondii ME49 [201]. Furthermore, the parasite was able to block the response of macrophages to LPS antigens [202], and also actively down-regulated of MHC class II molecules and was unable to up-regulate class I molecules in murine macrophages. These properties may represent an important strategy for evasion from the host’s immune response and for its intracellular survival [203].
Peroral T. gondii Infection Causes Development of Various Pathologic Changes in the Gastrointestinal Tract of both Experimental Animals and Humans
Animal studies
Development of GI tract inflammation following oral infection with T. gondii has been reported in a large number of different animal species belonging to two classes, mammalia and aves, and the most frequently affected organs were the liver (93.9% of the cases) and small intestine (57.6%) [105]. Additional sites affected with less frequency were the stomach, large intestines, and mesenteric lymph nodes. In most cases of experimental infections with the parasite, an inoculum of ≥102 and 40-100 tissue cysts of T. gondii type II and III (ME49 strain) were fed orally by the animals, and the small intestines (95%) and liver (85%) were the organs affected most frequently. In the majority of cases, small intestinal pathology was characterized by a complete loss of the villous architecture. In mice, the small intestines showed formation of edema between the epithelial layer and the lamina propria, secretion of fluid from the epithelial layer into the gut lumen, mild desquamation of epithelial cells, and moderate to severe necrosis [105]. Pathological abnormalities were most prominent in the distal part of the ileum, and intracellular parasites can be detected in large numbers in its lamina propria, while parasite numbers in the liver were smaller. It was suggested that the strain of T. gondii, the infectious inoculum, and the host genetic features affect development of intestinal pathology and severity of its course [105]. Specifically, in animals with acute and chronic infection peroral inoculation with various numbers of the parasite oocysts or tachyzoites caused significant changes in length, width and area of specific segments of the GI tract. In addition, it affected thickness of the intestinal wall, external muscle, mucosa, villous and enterocyte height, decreased numbers of various neurons in myenteric ganglions, and resulted in markedly changed proportions between the cell body, nucleus and cytoplasm of the intestinal neurons (Tables 9-13).
Table 9: Length, width and area of specific segments of the gastrointestinal tract in rats intraperitoneally infected with 106 tachyzoites of T. gondii type I (BTU IV strain) and examined 6 days post inoculation (acc. to Silva et al. [204]; with own modification).
Table 10: Length, width and area of the ileum-jejunum and the total colon in rats with acute and chronic T. gondii type III infection (acc. to Sugauara et al. [205]; with own modification).
Table 11: Thickness of the total wall, external muscle, mucosa, villous and enterocyte height in the jejunum of pigs orally infected with T. gondii type III (M7741 strain) and examined 30 and 60 days post inoculation (acc. to da Silva et al. [206]; with own modification).
Table 12: Mean ± SD of the number of NADPHd-p and NADHd-p neurons in 100 myenteric ganglions of the jejunum in pigs orally infected with oocysts of T. gondii type III (M7741 strain) and examined 30 days post inoculation (acc. to Odorizzi et al. [207]; with own modification).
Table 13: Median and P25, P75 percentiles of the cell body, nucleus and cytoplasm areas, and the nucleus/cell body ratio of NADH diaphorase-positive neurons in the jejunum of rats orally infected with 500 oocysts of T. gondii type III (M7741 strain) and examined 24 hrs post inoculation (acc. to Pereira et al. [208]; with own modification).
Alves et al. [209] showed that chronic subclinical infection of rats with T. gondii (confirmed by the presence of serum anti-T. gondii antibodies but no manifestations or signs of the infection were observed) resulted in a 14.73% decrease in the mean stomach weight when compared with control animals, and both the small and large gastric curvature regions of the glandular areas showed respectively 27.56% and 25.25% decrease of myenteric neurons population density (Table 14). Those authors [209] reported that no form of the parasite was been observed in the neurons studied. However, as the parasite needs nucleated cells to survive, one cannot exclude that at least some residues of T. gondii and/or its parasitophorus vacuole remained in the myenteric neurons because Pereira et al. [208] demonstrated that the only class of the rat jejunum neurons markedly decreased following oral inoculation with 500 oocysts of T. gondii type III (M7741) strain (examined 24 hrs after inoculation), was perikarion nerve cells between 151 and 200 μm2, thus large enough to eventually harbor various forms of the parasite, including parasitophorus vacuole and/or its remnants [210]. Enlarged the cell body, cytoplasm, and nucleus areas were also observed in the myenteric neurons of the ileum of rats acutely infected with the T. gondii type II (Table 15) [211]. On the other hand, Sugauara et al. [205] administered the parasite genotype III and found these cell bioparameters in the rat descending colon during acute infection decreased (Table 16), and Soares et al. [212] demonstrated augmentation of the cell body and cytoplasm areas in myenteric neurons of descending colon in rats chronically infected with T. gondii type I. Therefore, it seems that these quantitative and morphometric changes of the myenteric neurons are depending on acute or chronic infection, dose of inoculum required to induce intestinal pathology, genotype of the parasite used, and animal species infected [105]. Moreover, recent studies of Berenreiterova et al. [213] showed that the distribution of T. gondii cysts in the brain of a mouse with latent toxoplasmosis had no well targeted tropism although some brain regions were consistently more infected than others.
Table 14: Population density and the cell body area of myenteric neurons of glandular stomach in rats orally infected with 104 tachyzoites of T. gondii type III (BTU II strain) and examined 30 days post inoculation (acc. to Alves et al. [209];with own modification).
Table 15: Median and P25, P75 percentiles of the cell body, nucleus, and cytoplasm areas, and the nucleus/cell body area ratio of myenteric neurons in the terminal ileum and descending colon of rats orally infected with tissue cysts of T. gondii type II (ME-49 strain) and examined 24 hrs post inoculation (acc. to Sugauara et al. [211]; with own modification).
Table 16: Cell body, nucleus and cytoplasm areas, and nucleus/cell body area ratio of the myenteric neurons of terminal Ileum and descending colon in rats with acute and chronic infection with T. gondii type III (acc. to Sugauara et al. [205]; with own modification).
It must be noted that T. gondii genotype I strains are considered highly virulent in laboratory animals [214]. The T. gondii population in Europe and North America consists of three clonal strains, types I, II, and III [215]. However, in South America, Asia, and Africa, these clonal strains are less dominant, and recombinant and exotic strains are more common. Some of these atypical strains have been associated with the development of toxoplasmosis also in immunocompetent individuals [216,217]. In the Northern hemisphere genotype II and III strains have been presenting low virulence that promotes development of chronic infection with formation of tissue cysts in the central nervous system [215,218,219]. The virulence of genotype III Brazilian strains found in South America was different from those of the Northern hemisphere [220] and the strain M7741 isolated in the U.S. in 1958 was thought to have low virulence [221]. Recently, it was found that virulence of T. gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells [222]. During acute infection, effective presentation of parasite proteins to CD8+ T-ceIls appeared to be a consequence of active protein secretion by T. gondii and escape from the parasitophorus vacuole, rather than degradation of phagocytosed parasites or parasite’s bioproducts [223]. It must be added that type I strains replicate slightly faster than type II strains in vitro [217], and type I strains also have better migratory ability across biological barriers, including the lamina propria and submucosa [224]. Moreover, T. gondii type I strains interfere with NF-B activation and induce less IL-12 from infected macrophages in vitro than type II strains, suggesting that the parasite may be able to modulate the host immune response in a strainspecific manner [222,225,226]. Gut commensal bacteria also direct a protective immune response against the human pathogen T. gondii [227].
Studies performed in animals showed that oral infection with T. gondii resulted in different plastic changes in myenteric plexus neurons. For instance, in rats no changes in the population or density of these cells were observed [205,212,228], while there was an intense myenteric plexus neuronal cells death and atrophy reported in chicken [229]. On the other hand, in pigs orally infected for 30 days with oocysts of T. gondii III (M7741 strain) Odorizzi et al. [207] showed that the number of nitrergic NADPHd-p neurons per ganglion markedly increased, and the cells became hypertrophic through the augmentation of the cell body by 12.8% (P < 0.0001), and specifically through the increase of the nuclear area by 24.8% (P < 0.0001) (no change was observed in the distribution among different classes of neuronal cell sizes) (Tables 17 and 18). Because NO plays an important role in resistance of the host cells against T. gondii infection [230], it was suggested [207] that the cell hypertrophy may be indicative of the increased generation of biochemical mediator(s) with antiparasitic activity that may simultaneously exert a detrimental effect on tissue cells containing parasites [229,231]. Therefore, one may suggest that markedly different distribution of nitrergic neurons found in different ileal rat segments (Table 19) [232] is, at least in part, responsible for predominant dispersion of the gastrointestinal tract pathology and its intensity reported in both IBD and necrotizing enterocolitis in humans. Moreover, the marked decreases (and the recovery phase of CD8+ T cells) of T-lymphocyte subsets during proliferative response of mesenteric and splenic T lymphocytes demonstrated in mice orally infected with 40 cysts of the parasite strain 76 K (Table 20), as well as the variations in cytokine response profiles to diverse T. gondii antigens in the three different mouse strains studied (Tables 21-24) [233], may at least partly explain large variability of signs, symptomes and histopathologic changes observed during clinical course of IBD in humans. This reasoning is supported by the finding that TH1 and TH2 cytokines have opposing effects on gastrointestinal motility in gastrointestinal disorders via 5-HT signaling, i.e. TH1 cytokines downregulate CPI-17 (C-kinase potentiated protein phosphatase-1 inhibitor, m.w. = 17 kDa) and L-type Ca2+ channels and upregulate regulators of G protein signaling 4, which contributes to hypocontractibility of inflamed intestinal smooth muscles, and conversely, TH2 cytokines cause hypercontractibility via signal transducer and activator of transcription 6 or mitogen-activated protein kinase signaling pathways [234].
Table 17: Median and P25, P75 percentiles of the cell body, nucleus, and cytoplasm areas, and the nucleus/cell body area ratio of NADHd-p myenteric neurons of the jejunum in pigs orally infected with oocysts of T. gondii type III (M7741 strain) and examined 30 days post inoculation (acc. to Odorizzi et al. [207]; with own modification).
Table 18: Degree of correlation between the cell body, nucleus and cytoplasm areas of NADPHd-p and NADHd-p myenteric neurons of the jejunum in pigs orally infected with oocysts of T. gondii type III (M7741 strain) and examined 30 days post inoculation (acc. to Odorizzi et al. [207]; with own modification).
Table 19: Neuronal density (mean ± SD) in myenteric plexus of the mesenteric, intermediate and antimesenteric ileal segments in rats aged 7 months, estimated by different techniques of neuronal staining (acc. to Miranda-Neto et al. [232]; with own modification).
Table 20: Phenotypic analyses of the mesenteric and splenic T-lymphocyte subsets from C57BL/6 mice orally infected with 40 cysts of T. gondii strain 76K at the time of T-cell proliferative response (acc. to Chardes et al. [233]; with own modification).
Table 21: SAG1, GRA4 and 229-242 peptide-induced lymphocyte proliferation of mesenteric T cells from three inbred mouse strains orally infected with 40 cysts of T. gondii (76K strain) and collected on day 6 post inoculation. (acc. to Chardes et al. [233]; with own modification).
Table 22: SAG1, GRA4 and 229-242 peptide-induced lymphocyte proliferation of spleen T cells from three mouse strains orally infected with 40 cysts of T. gondii (76K strain), and collected on day 42 post inoculation. (acc. to Chardes et al. [233]; with own modification).
Table 23: Cytokine response profile of T. gondii antigen-specific mesenteric T lymphocytes from CBA/J mice orally infected with 40 cysts of T. gondii (76K strain) (acc. to Chardes et al. [233]; with own modification).
Table 24: Cytokine response profile of T. gondii antigen-specific spleen T lymphocytes from CBA/J mice orally infected with 40 cysts of the parasite strain 76K (acc. to Chardes et al. [233]; with own modification).
In rats infected with T. gondii genotype III (M7741 strain), morphometric analysis showed increased cell body and cytoplasm areas, and decreased nuclear area of the myenteric neurons [208]. The high degrees of the correlation value (Table 25) found between the subcellular structures of the jejunum neurons suggest that the observed hypertrophy of metabolically active neurons were related to the augmentation of the cytosol and/or organelles in the cytoplasm [208]. One cannot exclude that these cellular changes may be associated with the intracellular proliferation and metabolism of T. gondii tachyzoites/ bradyzoites/parasitophorus vacuole). Otherwise it was found that in the small intestine, the lymphoid tissues were represented by Peyer’s patches, which formed dome-like anatomic structures consisting of lymphocytes and dendritic cells [235]. The early Toxoplasma sonicateinduced mucosal T-cell proliferation occurred in the mesenteric lymph nodes and Payer’s patches with a peak responsivenes on day 6 post inoculation and rapidly reached background levels on day 7 post infection in Peyer’s patches and on day 8 post inoculation in mesenteric lymph nodes [233]. Subsequently, splenic cellular blastogenesis was observed from day 28 after infection and persisted throughout the experiment (day 91) [233].
Table 25: Degree of correlation between the cell body, cytoplasm, and nucleus areas of the jejunum neurons in rats orally infected with 500 oocysts of T. gondii type III (M7741 strain) and examined 24 hrs post inoculation (acc. to Pereira et al. [208]; with own modification).
After T. gondii infection, the host cells release a plethora of neuroimmune mediators that can seriously affect enteric nerves, including many cytokines and NO, to provide and regulate proper immune defense against the invading intracellular parasite [202]. Peroral T. gondii infection induced a significant decrease in the relative percentage of the T-cell population and the CD4/CD8 ratio, wheather it was on day 6 post inoculation with MLN cells (P < 0.01) or on days 28 and 56 post inoculation with splenic cells (P < 0.01 and P < 0.05, respectively) (Table 20) [233]. In the high responder CBA/J model, the mesenteric T-lymphocyte blastogenesis induced by Toxoplasma sonicate was associated with significant IFN-γ, IL-4, IL-5 and IL-6 production and little IL-2 secretion, whereas Toxoplasma sonicatespecific splenic T cells only generated IFN-γ, IL-6 and a higher level of IL-2. These findings suggested that mice orally infected with T. gondii induced a predominant mesenteric TH2-type cytokine response and a major spleen TH1-type response [233].
Finally, Bonapaz et al. [229] showed that chicken from the experimental group infected with oocysts of T. gondii type III (M7741 strain) and examined after 60 days had diarrhea, inflammatory infiltrates in the tunica mucosa, and duodenum layers atrophy (decreased thickness of all the studied bioparameters) (Table 26), as well as a marked increase in the number of caliciform cells. There was also about 70% loss of myenteric neurons, and the remaining cells presented a reduction of about 2.4% of the pericarion and 40.5% of the nucleus (P < 0.05) (Table 10). It was found that the intestinal mucosa goblet cells marked with PAS, which estimates glycoconjugates components of mucins and indicates a neutral mucus, increased 29.66% (P < 0.05) and the cells marked by AB, which estimates acid mucus, increased only 12.8% (P < 0.5). These changes suggested that the duodenal mucus became denser, thus providing protection intestinal epithelium from potential damage due to recurrent diarrhea [229] (Table 27). It must be noted that T. gondii sporozoites cross the intestinal mucosa either through the enterocytes or the caliciform cells [236], and the parasite was found in caliciform cells of the guinea pig conjunctiva minutes after inoculation [236,237].
Table 26: Thickness of the duodenal total wall, muscular and mucosal tunics, and muscularis mucosa in chicken orally infected with 1000 oocysts of the T. gondii type III (M7741 strain) and examined 60 days post inoculation (acc. to Bonapaz et al. [229]; with own modification).
Table 27: Number of goblets cells estimated by different histochemical techniques for glycoconjugates (components of mucins) in a 0.96 mm2 area of the duodenum tunica mucosa in chicken orally infected with 1000 oocysts of T. gondii type III (M7741 strain) and examined 60 days post inoculation (acc. to Bonapaz et al. [229]; with own modification).
Humans: In immunosupressed individuals, gastric abnormalities caused by T. gondii infection included ulcerations, thickening of gastric wall and folds, thickening and necrosis of tunica mucosa, as well as inflammatory infiltrates, were reported [238-240].
In patients with AIDS, T. gondii infection has been identified in the stomach, small intestine, colon, and esophagus in both biopsy specimens and during postmortem examinations [238,241,242]. Those patients, both men and women 22 to 39-yrs-old, presented with mildto- severe abdominal pain, nausea, vomiting, anorexia, weight loss, fever, and diarrhea. Endoscopic findings included ulcerated lesions and thickening of gastric folds and gastric wall, edema (the histologic correspondent of thickened gastric folds), focal necrosis, and sometimes narrowing of the antrum. The antrum and fundus appeared to be the main sites of involvement. The pathologic findings were variable acute and chronic cell infiltrates and the presence of tachyzoites, bradyzoites, and pseudocysts [242,243] identified in the lamina propria, endothelial cells, cytoplasm of the epithelial cells, and smooth muscle cells [243].
In AIDS patients with diarrhea and sometimes with lower GI tract bleeding [244-246] biopsy specimens of colonic/small intestine mucosa showed the presence of T. gondii tachyzoites, and endoscopy revealed multiple ulcers with raised margins [246,247].
Animals: T. gondii infection caused gastric lesions in several animal species [108], and inflammatory changes, necrosis of the gastric wall, as well as eosinophilic fibrosing gastritis [105,209,248-251]. Histopathologic, immunohistochemical and ultrastructural studies showed the parasite inside the epithelial, muscle, endothelial cells and macrophages [238], and in intracellular vacuoles in glandular cells on gastric wall [240]. Alves et al. [209] found that in rats T. gondii type III, tachyzoites were capable of resisting the gastric juice and crossed the GI tract barrier, reaching systemic circulation, because they documented the presence of anti-T. gondii antibodies in serum of the animals despite lack of clinical signs of the infection. There was a marked decrease in the population density of myenteric neurons of both the small and the large gastric curvatures (P < 0.012) with no change in the cell body area (Table 14). In addition, a decrease of the stomach area and its weight were also found [209]. Other authors also reported neuronal hypertrophy of duodenum and descending colon [205], whereas marked decreases in the nucleus and cell areas [205,253] occurred in the jejunum and ileum, respectively, demonstrating that myenteric plexus had been the subject of T. gondii infection [205,253-255]. It is interesting that no tissue cysts were found in the neurons of the gastric myenteric plexus evaluated by Alves et al. [209], but Montoya & Remington [241] suggested that these structures of the parasite have been diffusively and heterogeneously formed considering all tissues in the body. The lack of a blood-brain barrier and the absence of microglial cells in the ENS ganglions as compared with the central nervous system may result in augmented vulnerability of its neurons to T. gondii infection.
It was found that a single immunodominant surface antigen SAG1 exclusively expressed on the tachyzoite of T. gondii can elicit lethal inflammatory process in experimental model of pathogen-driven ileitis [256] through a robust B and T cell-specific response. SAG1 induces the dominant antibody response during infection [257,258] and a strong, systemic TH1-like T cell response characterized by high titer IFN-γ production by CD4 and CD8 lymphocytes [259]. Intestinal inflammation and tissue damage are distinguished by an exaggerated immune response mediated by both TNF-α and IFN-γ [260] and heightened sensitivity of intestinal epithelial cells to TNF-α [261-263]. IFN-γ is the cytokine mediator associated with intestinal inflammation following oral infection with T. gondii [255]. Moreover, recent studies showed that the parasite expresses serine and cysteine proteases that are critical for the assembly and trafficking of organellar content proteins [264], host cell invasion, replication and nutrient acquisition [265], finally at least in part affecting severity of the intestinal inflammation process.
In vitro stimulation of mesenteric T cells with three different T. gondii antigens resulted in secretion of IL-5 and IL-6 (except for SAG1) and IFN-γ, whereas no detectable IL-2 or IL-4 was observed (Table 23). It seems that mice orally infected with the parasite induced a predominant mesenteric TH2 cytokine response and a major spleen TH1 reaction [266], similarly as it was reported with other intestinal parasite infections [267,268]. Also miniature pigs fed with 1000 oocysts of the T. gondii VEG strain responded to the infection by simultaneously inducing proinflammatory cytokines, such as IFN-γ mRNA expression early post inoculation, and IL-12p35, as well as anti-inflammatory cytokine IL-10 later to provide a balance between controlling parasite growth and avoiding host pathology mediated by cytokine toxicity. In addition, a significant increase in the percentage of CD8+ cells was observed in the second week of infection [269].
Effect of Oral T. gondii Genotype III M7741 Strain Oocysts Infection in Rats on Nadph-Diaphorase Positive (Nitrergic) and Nadph-Diaphorase Negative (Cholinergic) Myenteric Neurons
Nitrergic neurons
The morphometric analysis of the NADPH-d+ (nitrergic) myenteric neurons performed by Hermes-Uliana et al. [270] showed atrophy for both the 30 DPI (19%) and 90 DPI (12.1%) rats as compared with control animals. However, the comparison of the neurons in the 30 DPI with those in the 90 DPI demonstrated hypertrophy in the latter (8.6%), which perhaps indicated a recovery phase of the cellular area (Table 28). At 30 DPI, an increase in the population of smaller (101-150 μm2) neurons (new young cells?) and a decrease in the population of the larger (201 to > 301 μm2) neurons (old, infected with the parasite and undergoing apoptosis cells?) were observed [270].
Table 28: Median and P25, P75 percentiles of the cell body, nucleus and cytoplasm areas, and the nucleus/cell body area ratio of the neurons stained with Giemsa and NADPHd-p in the jejunum of rats orally infected with 500 oocysts of T. gondii type III and examined 30 and 90 days post inoculation (acc. to Hermes-Uliana et al. [270]; with own modification).
The high degree of the correlation between the area of the cell body and cytoplasm in these cells subpopulation suggested that the morphological changes resulted mainly from the changes in the cytoplasm of those cells (r = 0.95, P < 0.001) [260]. It seems that rapid reduction of the excessive generation of NO in the inflamed intestinal tract tissue cells by mitochondria, and reversible inhibition of mitochondrial respiration by NO [271] may, at least in part, serve as an explanation of these subcellular changes.
After ingestion of contaminated food and water containing T. gondii cysts or sporulated oocysts [272], the bradyzoites released from the cysts first invade intestinal epithelial cells (IECs) leading to parasite dissemination throughout different organs. In response to the parasite infection, IECs and enterocytes released a plethora of cytotoxic mediators, such as for example NO, and up-regulated production of proinflammatory cytokines and chemokines such as MCP-1 (CCL2), MIP-2 (CXCL2), and IP (CXCL10), RANTES (CCL5), MCP-3 (CCL7), and CXCL9 [273-275]. Moreover, during T. gondii infection, it has been shown that commensals, such as E. coli, activated macrophages, dendritic cells and neutrophils through TLR4 engagement thus contributing to the parasite-associated intestinal inflammation [276]. Enteric bacteria were also found to induce cell death in porcine myenteric neurons via LPS, as a consequence of intestinal inflammation [277]. Experimental studies showed that bacteriotoxin LPS, acting via TLR4 activation and driving the TH1-type intestinal inflammatory process [278], caused delayed gastric emptying [279,280], intestinal dysmotility [280,281], and sphincter dysfunction [282], thus unraveling its damaging interaction with myenteric neurons [277]. It should be noted that the immune recognition by TLR4 also was involved in the protective mechanisms against peroral infection with T. gondii ME49 strain [201]. These are important findings because several investigations about the pathogenesis of inflammatory bowel disease adapted the view that the disease is due only to a fundamental abnormality in bacterial microflora of the gut [283].
Important Role Of Nitric Oxide During T. gondii Infection
The role of NO in parasitic diseases is very important [284]. Normally NO production is necessarily under tight control, but excessive NO can lead to development of immunopathology (diabetes, liver cirrhosis, rheumatoid arthritis). A number of cytokines, including IL-4, IL-10 and TGF-β, can downregulate induction of NO synthase in macrophages [284]. Also NO can reduce the activity of NO synthase by feedback inhibition, and inhibits the production of IFN-γ by TH1 cells with the regulatory pathways involving tyrosine kinase and protein kinase C [284]. NO (and IFN-γ) plays an important role also in upregulation of VEGF gene expression [285], the factor known to be markedly increased in the cerebrospinal fluid of patients with ASD [36].
NO is a cytotoxic effector molecule produced by macrophages [286] that acts by inhibiting essential mitochondrial and nuclear enzymes [287], and results in iron mobilization from tumor target cells, which inhibits DNA synthesis and mitochondrial respiration. Mitochondria may contain NO synthase and can produce significant amounts of NO to regulate their own respiration. This function may therefore be important for physiological and pathological (because of a known overproduction of NO in autism) regulation of energy metabolism [288-290] in gastrointestinal tract.
NO is known to affect the development and function of the central nervous system, such as neurite growth [291], synaptogenesis [292], neurotransmitter release [293], memory processing and learning (learning was dose-dependently affected) [294, 295], and macrophagemediated cytotoxicity [286]. The expression of iNOS and production of NO also affect inflammatory processes [296], for example peroxynitrite (a product of NO and superoxide anion) generated by iNOS synthase and NADPH oxidase mediated microglial toxicity to oligodendrocytes [297], and NO exerted a dose-dependent biphasic regulatory effect on the activity of matrix metalloproteinase-9 secreted from murine macrophages [298]. Recently, it was reported that human enteric glial cells directly responded to proinflammatory stimuli by changing their expression profile and by proliferating, and were able to increase production of NO through release of glial proteins, such as S100B [299,300]. S100B is specifically and physiologically expressed by enteric glial cells and its overexpression is associated with the onset and maintenance of intestinal inflammation because it has a proinflammatory activity, which gain access to the extracellular space especially at immune-inflammatory reaction sites in the gut [66]. NO also modulated T-lymphocyte migration in Peyer’s patches and in nonlymphoid villous submucosa of rat small intestine through a significantly increased rolling and adherence of lymphocytes in postcapillary venules of Peyer’s patches and submucosal venules without markedly decreasing red blood cell velocity [301]. Thus, T. gondii infection may be, at least in part, responsible for development of gastrointestinal symptoms and intestinal entropathy, including the characteristic ileo-colonic LNH, increased enterocyte and Paneth cell numbers reported in autistic children [3,6,7,25,302]. The increased number of cells per ganglion, hypertrophy of NADPH diaphorasepositive (nitrergic) neurons, the decrease of the cellular number, as well as their nuclear area in these nitrergic (metabolically more active) neurons [207,208,270], and the significant increase of the villous and enterocyte height [206] found in the laboratory animals following oral inoculation with the parasite, are in agreement with the above-presented reasoning. It must be noted that in mice infected by the VEG strain of T. gondii, great amounts of parasites were found in the mesenteric lymph node, 48 hrs post infection [303]. In this context, the ileo-colonic LNH observed in some autistic children may therefore reflect an enhanced immune response [26] to chronic phase of oral T. gondii infection. This may be supported by the fact that lactoferrin administered orally improved gastrointestinal morphology in growing calves because it enhanced size of Peyer’s patches in the ileum and decreased villous size in the jejunum [304]. Moreover, these changes are in line with the intestinal morphometric abnormalities reported in animals orally infected with oocysts of T. gondii type II or type III strains [205,206,208,211,270]. In the experimental animals, PCR analysis showed the presence of T. gondii only in the mucosa and submucosa, which may indicate that the finding in the myenteric plexus occurred as a result of an indirect action of the parasite via cytokines and other immune biomediators released [305,306]. Myenteric neurons are target cells for paracrine secretions from the immunocytes at the intestinal wall [307] or even the myenteric ganglions themselves [173] because neurons within this plexus are closely associated with the immune system [308]. It must be added that mast cell-nerve interactions play a key role in intestinal inflammation because mast cells secrete chemotactic factors able to recruite neutrophils, macrophages and lymphocytes when the parasite reaches the lamina propria [309], finally reducing survival of myenteric neurons [310].
The genotype II strains of T. gondii have low virulence in mice and high cystogenic capacity and the positive serologic anti-T. gondii titers found by Hermes-Uliana et al. [270] ensured that the animals have been successfully infected with the parasite. However, the authors did not find tissue cysts of the parasite in the myenteric neuron samples evaluated and this is in agreement with the observation of Dubey et al. [272] that intestinal cysts are rarely found. In addition, the maintenance of the same number of neurons reported by Hermes-Uliana et al. [270] was similar to the findings reported by other authors [205,211,212] in the small and large intestine of animals infected orally with T. gondii tachyzoites or bradyzoites. Moreover, the myenteric neurons of the total population became atrophic in the 30 DPI animals and experienced recovery followed by hypertrophy 90 DPI, independently of the class studied. Both, the atrophy and hypertrophy of these cells resulted mainly from the changes in cytoplasmic area, and the area of nucleus changed proportionally to these alterations [270]. Preliminary studies in rats showed atrophy in the myenteric neurons of the ileum 30 DPI after infection with tachyzoites from another genotype II strain [211]. In contrast, T. gondii genotype III [205] and genotype I infection [212] caused neuronal hypertrophy in the large intestine of rats 30 DPI. Thus, it seems that changes in enteric neurons found after peroral infection of animals with T. gondii depend on the parasite genotype used, infecting material and its virulence/antigenicity, part of gastrointestinal tract infected, animal species used, and their innate immunity.
Cholinergic neurons
After T. gondii infection, the number of cholinergic neurons in rats showed no changes in the population density and remained as the predominant subpopulation of the cells in comparison with the nitrergic neurons [270,311], in spite of the fact that cholinergic neurons are considered vulnerable to neuronal death [312]. This finding may be important for controlling intensity of intestinal inflammation in the animals infected with the parasite because in mice cholinergic stimulation of macrophages reduced their production of TNF-α through a mechanism involving nicotinic receptor activation [313]. It must be noted that in the cortex of autistic patients, the cholinergic receptors known to be sensitive to NO toxicity were found to be decreased [314]. However, it was found that the treatment with cholinergic agonists improved behavioral abnormalities in those individuals [315,316]. These beneficial therapeutic effects may be explained by the finding that proinflammatory cytokine levels and excessive inflammation can be regulated by specifically augmenting cholinergic signaling via the efferent vagus nerve and/or applying selective cholinergic modalities targeting the 7 subunit-containing nicotinic acetylcholine receptor [317,318].
Gastrointestinal Tract Disturbances in Autistic Patients May Be At Least in Part Caused By Mast Cell Activation During Peroral T. gondii Infection Associated with Reduced Surivival of Myenteric Neurons
Several children with ASD suffer from “allergic-like” symptoms [319-321], and many of them may be consistent with chronic idiopathic or chronic autoimmune urticaria [322]. A case-control study of Croen et al. [323] showed that prevalence of maternal psoriasis, asthma, hay fever and atopic dermatitis during the second trimester of pregnancy correlated with > 3-fold elevated risk of ASD development in their children. Recently, a strong link between allergies and autism has been also suggested [324].
Inflammatory cells release an array of cytokines and inflammatory mediators that affect enteric nervous system. Mast cells are ascribed a central role in mediating hyperexcitability and neuron depolarization via their release of histamine, prostaglandins, leukotrienes and tryptase [325,326]. The elusive mast cells increase in number in several gastrointestinal disorders, including infectious diseases, food allergy, IBD, and IBS [310,327]. Crohn’s disease associated dysmotility has been attributed to fibrosis and damage to enteric nerves and interstitial cells of Cajal (ICC) [137]. Ultrastructural injury to ICC was patchy and ICC-Auerbach’s plexus showed damage more frequently, i.e. swelling of mitochondria, decreased electron density, autophagosomes and partial depletion of cytoplasm. Electron microscopy showed mast cells exhibiting piecemeal degranulation and making frequent and selective membrane-to-membrane contact with all types of injured ICC, which suggested chronic release of granule content [137]. Nb. it must be added that mercury used as a preservative in vaccines also induced inflammatory mediators release from human mast cells [328], and recently DeLong [329] demonstrated a positive and statistically significant relationship between the proportion of children who received the recommended vaccines by age of 2 years and the prevalence of autism or speech/language impairment across the U.S. population.
In pigs orally infected with oocysts of T. gondii type III (M7741 strain), Silva et al. [206] demonstrated hypertrophy of the intestinal wall (the increases in the villous and enterocyte heights, mucosal and external intestinal muscle thickness) of jejunum 60 days post inoculation. It must be noted that at 30 days post inoculation, there was atrophy of the intestinal wall with a decrease in the external muscle and increase in villous height [206]. Chickens infected with oocysts of the same strain of the parasite also showed mucosal atrophy in both the duodenum [330] and ileum [331]. Mice fed with oocysts of T. gondii VEG strain had great amounts of parasites in the mesenteric lymph nodes [331]. The studies of Silva et al. [204] in rats intraperitoneally infected with 106 tachyzoites of T. gondii type I (BTUIV strain) showed that approximately 14.9% of the myenteric neurons in the jejunum died (P < 0.05) and the cellular response of myenteric neurons to the infection was heterogenous depending on the small and large intestinal segment (Table 29). In the neurons of control animals jejunum, the nucleus occupied 52% of the cell body and the infection caused its reduction to 48% (P < 0.05) (Table 30), which suggested decreased nuclear [204] and increased cytoplasmic metabolism. In the jejunum of pigs orally inoculated with oocysts of T. gondii type III (M7741 strain) there was also an increase in the cytoplasmic area in NADH-diaphorase positive neurons that are considered more metabolically active, while the area of the cell body did not change markedly (Table 17) [207]. The morphometric alterations in the intestine wall probably have been caused by the proliferation and/or higher recruitment of immune cells to the lamina propria as a result of T. gondii invasion because, for example, mast cells activation results in secretion of chemotactic factors that attract neutrophils, macrophages and lymphocytes [333,334]. It was found that the mast cells found in the ileum submucosa 48 hrs post intraperitoneal inoculation with 105 tachyzoites contained T. gondii (Table 31) and also showed significant morphological alterations, including an increase in the amount of their cytoplasmic projections in the contact area with the parasites, as well as cytoplasmic granules with flocculent material and electron lucid halo, granule fusions, outlining wide intracytoplasmic channels, possibly due to the intense degranulation process, probably in response to the discharge of the granule contents from mast cells, such as cytotoxic leukotrienes on the parasites present in this compartment [333,335]. Mast cells were found to interact with other parasitized cells, including macrophages. [Nb. it must be noted that proliferation rate of intracellular T. gondii tachyzoites varies depending on the cell type affected (Table 32)]. In experimental animals free tachyzoites with morphological alterations and parasitophorus vacuoles inside mast cells were demonstrated within one hour after intraperitoneal inoculation [333]. These findings may be responsible for the enteroglial-sustained intestinal inflammation in IBD because they are similar to the perpetuum mobile-like biomachinery probably responsible for the persistent neuroinflammation in patients with autism and other neurodegenerative diseases recently proposed by Prandota [210], in which host-endoplasmic reticulum-parasitophorus vacuole interaction provides a route of entry for antigen crosspresentation in T. gondii-infected dendritic cells [15].
Table 29: Neuron population density per 1 cm2 of specific segments of the gastrointestinal tract and projection of these numbers to the total area of each segment of intestine in rats intraperitoneally infected with 106 tachyzoites of T. gondii type I (BTU IV strain) and examined 6 days post inoculation (acc. to Silva et al. [204]; with own modification).
Table 30: The cell body, nucleus and cytoplasm areas, and the nucleus/cell body area ratio of myenteric neurons of specific segments of the gastrointestinal tract in rats intraperitoneally infected with 106 tachyzoites of T. gondii type I (BTU IV strain) and examined 6 days post inoculation (acc. to Silva et al. [204]; with own modification).
Table 31: Relative numbers of lymphocytes, macrophages, mast cells and neutrophils from peritoneal cavity of the wild mouse Calomys callosus intraperitoneally infected with 105 tachyzoites of T. gondii RH strain, from 1 to 48 hrs post inoculation, when compared with uninfected animals (acc. to Ferreira et al. [333]; with own modification).
Table 32: Division rate of intracellular T. gondii tachyzoites in primary human cells in vitro (acc. to Channon et al. [336]; with own modification).
Important role of mast cells in gastrointestinal inflammation
Mast cells are important in allergy and inflammation [321,353] because they contain histamine, proteoglycans, and neutral proteases in various compositions. Lipid-derived mediators include the metabolites of arachidonic acid by cyclooxygenase and lipogenase, various cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-16, TNF-α, TNF-β) and chemokines (MIP-1, MCP-1) are synthesized and secreted early after contact with antigen [354]. Mast cells play also a regulatory role in the innate and acquired immune responses because they influence T and B cell functions, present antigen to T cells, and can directly stimulate IgE synthesis in B cells [354]. Mast cells are attracted to the molecules secreted by the enterocytes in the basolateral region of the intestinal crypts [206,275], finally inducing NF-κB, which plays a key role in the regulation of the inflammatory, immune and antiapoptotic response in the host infected with T. gondii [355]. Interestingly, on the basolateral enterocyte surface of autistic children with gastrointestinal disturbances, deposition of IgG1 and IgG4, co-localizing with complement C1q, was shown to be accumulated compared to healthy controls [7]. It must be noted that T. gondii penetrates intestinal epithelial cells, and either develops within them, or exits through the basolateral side and burrows across the basement membrane [224,356]. Buzoni-Gatel et al. [357] showed that the intensity of experimental ileitis after infection with the parasite was controlled by TGF-β-producing intraepithelial lymphocytes (IELs), since primed IELs reduced production of inflammatory chemokines by the infected enterocytes, and IFN-γ by splenocytes. Finally, a striking mast cell-induced myenteric neuronal cell death in culture (probably mediated via PAR2 activation, IL-6 and prostaglandin D2) was demonstrated [310].
Serum Antineutrophil Cytoplasmic Autoantibodies (ANCA) in Patients with Inflammatory Bowel Disease (IBD) may be Generated by Chronic Oral T. gondii Infection
Lidar et al. [358] demonstrated that titers of anti-IgM (7.5 vs. 1%) and anti-IgG (33 vs. 26.9%) antibodies toward T. gondii, hepatitis C virus (HCV) and Saccharomyces cerevisiae were markedly higher in sera of IBD patients (80 with CD, 39 with UC) than in controls (98 healthy individuals). The higher prevalence of IgM antibodies resulted from a significantly increased frequency in CD patients (13.5 vs. 1%, P < 0.001). They suggested that whereas an excess of anti-HCV may be the result of immunosuppression from the inflammatory disease, the parasite has an initiating role in the etiopathogenesis of human IBD, especially CD, thus corroborating the murine model [276,358]. Although Lidar et al [358] did not cite or discuss this finding in their later publications, they believed that certain infections via for example molecular mimicry could generate an immunological environment that confers protection from certain autoimmune conditions, such as CD [358,359]. Most recently, Egan et al. [360] also proposed that T. gondii acts as a trigger setting into motion a series of events culminating in loss of tolerance in the intestine and emergence of pathogenic T cell effectors. This suggestion was based on the finding that oral infection of certain inbred mouse strains with the parasite caused inflammatory pathology resembling lesions seen during human IBD, in particular CD [360].
Perinuclear ANCA has been found consistently in serum of children and adults with IBD, and occurs significantly more often in UC than in CD [361-365]. The presence of ANCA may be associated with peroral T. gondii infection of those individuals because Rattan et al. [106] demonstrated a higher incidence of positive Sabin-Feldman dye test to the parasite among patients with Crohn’s disease over the age of 40 (P < 0.05). This finding is in line with the increased seropositivity to T. gondii infection along with age reported by Hinze-Selch et al. [366] (Table 33). [Nb. the seroprevalence of anti-Toxoplasma IgG antibodies and IgG titers was found to be significantly higher in patients with abdominal hernia repair than those without hernia, and in ≥ 50 years old individuals than those < 50 yrs old [367]. This is also in agreement with the above-presented reasoning that the parasite causes damage to myenteric neurons and markedly affects the cell body, nucleus and cytoplasm areas and nucleus/cell body ratios depending on acute or chronic infection (Tables 13-18,25,28,30). It should be noted that the parasite has the ability to infect every type of nucleated cell in mammals, including neutrophils, and Ferreira et al. [367] demonstrated a remarkable increase in the influx of neutrophils toward the peritoneal cavity of infected experimental animals already 12 hrs post infection (Table 31). It was reported that mast cell-deficient mice displayed a significant defective ability to recruit PMNs during early infection by T. gondii, which suggested that these cells served as a major effector cell type involved in neutrophil recruitment, and CXCR2 was required for this process [368]. Finally, recently Prandota [210] suggested that the increased generation of antibodies directed against brain proteins in patients with autism and their families may be caused by T. gondii infection and emphasized possible important role of maternal and fetal microchimerisms in these processes.
Table 33: Percentage of T. gondii positive individuals among 214 nonpsychiatrically affected controls depending on age analyzed during a large epidemiologic study of 869 psychiatric patients [366]).
Table 34: Hypoxia-inducible gene expression [380].
Prenatal and Postnatal Oral T. gondii Infection May Cause Development of Necrotizing Enterocolitis and Inflammatory Bowel Disease in Newborns, Children and Adults
Necrotizing enterocolitis (NEC)
NEC, an inflammatory disease of the terminal ileum, cecum, and ascending colon, is the most common gastrointestinal disease of infancy, afflicting 5-15% of all infants born at less than 30 weeks gestational age or < 1500 gm birth weight. However, up to 10% of all neonates who develop NEC are born at term [369,370]. NEC occurs principally in premature infants after the introduction of artificial oral feeding [371]. Pathophysiology of NEC in term infants is often associated with congenital heart disease, a recent “bypass” operation, hypoxemic-ischemic events, polycythemia [115,372]. There is also strong evidence that the initial bacterial colonization of the newborn intestine plays a pivotal role in the development of NEC [114,373,374]. Pathologic microorganisms, particularly Gram-negative bacteria, such as, Enterobacter, Enterococcus Clostridia, and Staphylococcus, are predominant fecal bacterial species with very little colonization with Bifidobacteria [115,375]. LPS interaction with enterocytes via TLR-4 involves activation of NF-κB to stimulate generation of various proinflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-8, IL-10, IL- 12, IL-18 [115], and for instance, IL-8 stimulates migration of neutrophils from intravascular to interstitial and luminal sites [374]. It was found that immature human enterocytes react with excessive proinflammatory cytokine production, and IL-8 activity was noted predominantly in villous and crypt epithelium but also in a few immunoresponsive lymphoid cells [376]. In addition, there was a pronounced TLR-4 expression documented in fetal human intestine [377] and in formulafed and hypoxia-stressed rats [115,378]. In this context, it must be emphasized that T. gondii activates hypoxia-inducible factor 1 (HIF1) already at physiologically relevant oxygen levels and requires HIF1 for growth and survival [379], and HIF1 activates gene expression of several proinflammatory cytokines (including NO synthase-2), various enzymes, and biologic substances (e.g. erythropoietin) (Table 34). [Nb. erythropoietin is a breast milk and amniotic fluid component that plays an important role in intestinal development, cell migration, and intestinal restitution [381,382].] It must be noted that hypoxia as a risk factor of NEC development and its intensity is in line with recent suggestion that migraine associated with patent foramen ovale may be caused by reactivation of cerebral toxoplasmosis triggered by arterial blood oxygen desaturation [380], and also with hypoxemic-ischemic events associated with clinical course of other congenital heart diseases [372]. Finally, hypoxia may also impair function of the CO2, aquaporin 1 (AQP1), and AQP4 gas channels, thus enhancing neuroinflammation in children with NEC, especially those with RhAG phenotype of the red blood cells, as it was suggested in autistics [383]. Recently, in the U.S. Olariu et al. [384] reviewed clinical data and laboratory profiles of 164 infants with congenital toxoplasmosis whose mothers had not been treated for the parasite during gestation and found that 84% of the infants had one or more severe clinical manifestations, including eye disease (92.2%), brain calcifications (79.6%), and hydrocephalus (67.7%). In 61.6% of the infants, these abnormalities were present concurrently. It appeared that T. gondii-specific IgM, IgA, and IgE antibodies were demonstrable in 86.6%, 77.4%, and 40.2% of the infants, respectively. The authors emphasized that these results contrast remarkably with several European investigators who rarely observe severe clinical signs in infants with congenital toxoplasmosis [384], and one cannot exclude that T. gondii genotype as well as various diagnostic methods used estimating only certain parasite antigens may be at least in part responsible for the difference (Tables 35-37). It is now apparent that many atypical genotypes exists besides the typical 3 genotypes that can differ in pathogenicity and transmissibility from the typical genotypes used so far in scientific research, thus changing paradigm of congenital toxoplasmosis [388].
Table 35: Clinical characteristics of fetus and newborns with T. gondii infection, and relationship with the main genotypes of the parasite (acc. to Ajzenberg et al. [385]; with own modification).
Table 36: Congenital transmission of T. gondii in humans. (acc. to Hide et al. [386]; with own modification).
Table 37: Risk of T. gondii vertical transmission and frequency of morphological abnormalities of fetus with increasing gestational age [387].
Table 38: Myenteric neuron population density in the four regions of the cecum and the entire organ of control rats (CG) and rats inoculated with the genotype I (EG1) and genotype III (EG2) strains of T. gondii (mean ± SD) (acc. to Zaniolo et al. [428]; with own modification).
Ajzenberg et al. [385] studied the influence of T. gondii genotypes on the severity of human congenital toxoplasmosis (asymptomatic, benign, or severe infection of newborn or fetal death) and showed that type II isolates were largely predominant (84.88% of 74 different genotypes and 96.49% in 57 consecutive cases) (Table 35). Type I and atypical isolates were not found in asymptomatic or benign congenital toxoplasmosis, and in 4 cases T. gondii was isolated from placenta [385], which may suggest that these children after all were infected with the parasite via maternal microchimerism.
Development of NEC may be due to prenatal or postnatal peroral infection with the parasite because, for example Bonapaz et al. [229] showed that chicken infected with oocysts of T. gondii type III (M7741 strain) and examined after 60 days had diarrhea, inflammatory infiltrates in the tunica mucosa, and duodenum layers atrophy (decreased thickness of all the studied bioparameters) (Table 26), as well as about 70% loss of intestinal myenteric plexus neurons. These alterations of the intestinal wall and myenteric plexus of hen duodenum leading to diarrhea irreversibly impair acquisition of nutrients and gastrointestinal tract motility, finally resulting in death of animals. One cannot exclude that similar sequence of pathophysiological events plays an important role also during development of NEC in young infants. This reasoning may be supported by the morphologic alterations of the ENS and deficiency of non-adrenergic non-cholinergic (NANC) inhibitory innervation reported in neonatal NEC [389,390]. The deficiency in NANC inhibitory innervation may contribute to the formation of functional obstructions following acute NEC [389]. Examination of the myenteric plexus and external submucosal plexus by whole-mount immunohistochemistry revealed a marked reduction in glial cells concomitant with gradual deterioration of nerve cells, both abnormalities predominating in antimesenteric intestinal circumference, where ischemic lesions tended to appear first [390]. The most severe damage of nervous tissue was found in the plexus mucosus and submucosus internus. The destroyed ganglia appeared like “empty baskets” (residual tangles) and housed both glial and nerve cells with various stages of cell deterioration and the formation of central lesions within the myenteric ganglia [389,390]. It must be noted that neurons of the ENS are intimately associated with enteric glial cells that share many similarities with astrocytes of the CNS, and acute loss of enteric gial cells induces massive pathological changes with similarities to NEC and early Crohn’s disease [391]. In this context, it must be emphasized that reduction of Purkinje cell size (by 24-50%), number, and cerebellar volume, particularly of the posterior lobe, was reported in the patients with autism [392,393]. Recently, Arndt et al. [394] suggested that these findings may be explained by the finding that the Purkinje cell body is wrapped by processes of the neighboring basket cells therefore the late loss of Purkinje cells is characterized by the presence of “empty baskets”. Prenatal exposure of rats to valproic acid also reproduced the cerebellar anomalies associated with autism [395]. Interestingly, gastrointestinal symptoms, such as nausea, vomiting, dysphagia, constipation, diarrhea, and heartburn, were highly common adverse effects of valproate in intractable epileptic patients treated with valproate for a long-time [396,397].
Finally, pathognomic of NEC pneumatosis intestinalis creating characteristic indentations sometimes seen radiographically and endoscopically [398,399] may be caused by heme oxygenase-1 system activation leading to generation of CO, which plays an important protective role during acute and chronic gastrointestinal inflammation and oxidative injury [400-402]. Otterbein et al. [401] demonstrated that CO inhibits production of proinflammatory cytokines (TNF-α, IL-1β, macrophage inflammatory protein-1β) and stimulates synthesis of the antiinflammatory cytokine IL-10.
Cytomegalovirus-Associated Nec and Other Gastrointestinal Tract Abnormalities May Be Rather Caused by T. gondii Infection Comorbidity
Gastrointestinal manifestations of post-natally acquired cytomegalovirus (CMV) in preterm neonates can vary from mild diarrhea to severe NEC. CMV has been detected in breast milk in 32 to 96% of CMV seropositive mothers and the rate of transmission was 37% [403]. Gessler et al. [404] reported a development of NEC in a preterm twin after breastfeeding and his CMV serology tests determined at the 5th, 13th, and 27th week of gestation were IgG 85, 92, ad 94 U/ml (positive), respectively, and the IgM negative. However, the child was also Toxoplasma-IgG seropositive (IgG 39 U/ml, IgM negative), which may suggest chronic infection. CMV was detected also during acute NEC [405], as well as during the proliferative stage of stricture development after NEC [406-408], and ileal perforations and gastrointestinal beeding have been reported even in elderly immunocompetent individuals [409-413]. The clinical and endoscopic features of 20 cases with gastrointestinal CMV disease showed that many patients were immunocompromised, and the endoscopic abnormalities involved inflammatory mucosa alone (3/20), ulceration alone (7/20), inflammatory mucosa associated with ulcer (9/20), and submucosal tumor with ulcer (1/20), and the most common abnormality were multiple ulcers with at least one large ulcer [414]. Rafailidis et al. [415] analyzed 89 articles reporting on severe CMV infection in 290 apparently immunocompetent adults and found that the gastrointestinal tract (colitis) and the CNS (meningitis, encephalitis, and transverse myelitis) were the most frequent sites of infection. Constant seroprevalence of T. gondii and CMV infection reported among pregnant women (anti-Toxoplasma IgG antibody vs anti-CMV IgG, 48.3%-69.3% vs. 76.6%-96.4%, respectively) [416-418], as well as the common comorbid pathology of HIV-infected patients (CMV infection vs cerebral toxoplasmosis, 10.1% vs. 9.2%, respectively [419], may suggest that the gastrointestinal abnormalities found in CMV seropositive children and adults were at least in part due to concomitant T. gondii infection.
Chronic Constipation may be due to Post-Inflammatory Damage to the Enteric Nervous System by Prenatal or Postanatal Oral T. gondii Infection
Chronic constipation with megarectum is frequently reported in children with autism [420]. It is known that malformations or lesions of the ENS may lead to a severely prolonged intestinal transit time resulting in chronic constipation resistant to conservative treatment. The most recognized intestinal innervation disorder
represented aganglionosis (Hirschsprung’s disease) characterized by the absence of intramural nerve cells and the hypertrophy of the nerve fiber bundles within the affected intestinal segment [421]. Nonaganglionic intestinal innervation abnormalities include intestinal neuronal dysplasia (IND), hypoganglionosis and heterotopic ganglia [422]. At present, the pathogenesis of intestinal neuronal malformations is mainly attributed to developmental disorders of the ENS, in part caused by genetic defects, and until now no unified pathophysiological concept has been established to explain the ENS sustained damage during the postnatal period [421]. Histologic study of Tomita et al. [423] performed in children (aged 2-15 years) with chronic refractory constipation, such as Hirschsprung’s disease, hypoganglionosis, and IND, showed that the incidence of hypoganglionosis was significantly greater than that of hypoganglionosis and IND (P < 0.01, P < 0.001, respectively). Both these entities could be diagnosed by full-thickness rectal mucosal biopsies, especially by NADPH-diaphorase and acetylcholinesterase staining [423,424]. Wester et al. [425] investigated specimens obtained from postmortem small bowel and colon in 20 children (aged one day to 15 years) with IND and found that the density of ganglion cells in the myenteric plexus decreased significantly with age during the first 3-4 years of life at all levels of small bowel, colon, and rectum. The meshwork of fibers also becomes sparser during that time. The histological features of IND are mainly confined to the submucosus plexus [425]. However, Meier-Ruge [426] also found hyperplasia of the myenteric plexus, and rarely, isolated hyperganglionosis of the myenteric plexus with no submucous abnormalities has been described [427]. It appeared also that the NADPH diaphorase positive (nitrergic) subpopulation represented about 34% of all neurones in the myenteric plexus [425]. In adult patients with slow-transit constipation and megacolon, the intramuscular networks of the interstitial cells of Cajal showed a significantly reduced density (nb. interstitial cells of Cajal contribute markedly to the mediation of intestinal motility by generating “slow wave” activity) [424]. All these marked postnatal alterations in the myenteric plexus, and huge variations between these reports analyzed by Wester et al. [425] may be in part explained by various T. gondii genotypes and numbers of oocysts/sporozoites/bradyzoites/tachyzoites affecting different parts of the gastrointestinal tract of patients with IND. This suggestion is supported by the morphological changes in the cecal myenteric neurons depending on the genotype I (BTU IV) or III (BTU II) T. gondii strains [428]. The parasite dose of 105 tachyzoites administered orally in rats tested later positive for the presence of IgG against T. gondii caused different effects on the population density and morphometry of myenteric neurons within the four regions of the intestine investigated (Tables 38-41) [428]. Moreover, it was found that although T. gondii RH and Ankara strains had dense protein bands between 60 and 70 kDa and at 15 kDa, TS-4 strain had different and fewer bands [429]. This may at least partly serve as an explanation for the differences in molecular weights of antibodies and autoantibodies against brain proteins reported in various autistic individuals [210].
Table 39: Nuclear area of myenteric neurons in the four regions of the cecum and the entire organ of control rats (CG) and rats inoculated with the genotype I (EG1) and genotype III (EG2) strains of T. gondii (median and percentiles 25; 75) (acc. to Zaniolo et al. [428]; with own modification).
Table 40: Cytoplasmic area of myenteric neurons in the four regions of the cecum and the entire organ of control rats (CG) and rats inoculated with the genotype I (EG1) and genotype III (EG2) strains of T. gondii (median and percentiles 25; 75) (acc. to Zaniolo et al. [428]; with own modification).
Table 41: Cell body area of myenteric neurons in the four regions of the cecum and the entire organ of control rats (CG) and rats inoculated with the genotype I (EG1) and genotype III (EG2) strains of T. gondii (median and percentiles 25; 75) (acc. to Zaniolo et al. [428]; with own modification).
Headache, Abdominal Migraine, Recurrent Abdominal Pains and Development of IBD
A link between recurrent headache/migraine and gastrointestinal disorders has been confirmed by many clinical observations and epidemiologic studies [430-434].
Abdominal migraine (AM) occurs in 1-4% of children and represents 4-15% of pediatric gastroenterology patients followed for idiopathic non-colicky abdominal pain [435-437]. Pallor, anorexia, nausea, vomiting, photophobia, or headache may be associated with the episodes, and a family history of migraine headaches often is noted. AM emerges between the ages of 3 and 10 years [438], and evidence suggests its evolution into migraine headaches, being a “precursor of migraine” [439]. Carson et al. [437] found that it was 4 times more likely for a patient with recurrent abdominal pain to have AM if they also had migraine headache (P < 0.024), and an esophagoduodenoscopy performed as part of their diagnostic evaluation (P < 0.008). Frequently reported clinical features among suspected AM patients included nausea (P < 0.001), vomiting (P < 0.038), and anorexia (P < 0.001). The nonabdominal pain has been reported as periumbilical (63% of patients), midline (11%), and poorly localized (5%) [438]. In preschoolage children recurrent abdominal pain epidemiology showed a prevalence ranging from 0.3% to 19% (median 8.4%) higher in females [441,442].
Endoscopic examination performed in a consecutive series of 31 children (median age 12 yrs) with recurrent abdominal pain and suffering from migraine with (n = 21) or without (n = 10) aura demonstrated that 41.9% of them had esophagitis, 51.6% - gastritis of corpus, 38.7% - antral gastritis, and 87.1% had duodenitis [432].
In one neurologic study, functional gastrointestinal disorders were reported in 69% of patients with migraine in periods between the attacks (eg. diarrhea in 16-20%), and specifically IBS in 43% of them [443]. Watson et al. [430] found that frequent headaches were noted by 50% of 90 patients with IBS and only 18% of controls (447) (P < 0.001). In the IBS group, the headaches were age-related, the prevalence being greatest (65%) in women 16 to 30 years of age and diminishing with advancing age. Investigations based on a questionnaire performed in general population (1620 persons) showed that migraine was present in 32% (112 questioned individuals) of patients with IBS compared with 18% (229) (P < 0.01) of those without the syndrome [431].
Finally, it must be noted that the recurrent headache/migraine attacks have been reported in several infants, children, adolescents, and adults with markedly increased serum anti-T. gondii IgG antibody levels, and pharmacological treatment directed against the parasite was successful in those individuals [444-446]. These findings may be indirectly supported by the fact that intravenous valproic acid, a drug used for migraine prophylaxis, has been on occasion administered with apparent success in cases of abdominal migraine [447-449], and in vitro studies showed its high activity against T. gondii (Table 42) [450].
Table 42: Drugs tested for in vitro activity against T. gondii (acc. to Jones-Brando et al. [450]; with own modification).
Development of Metabolic Syndrome in Patients With IBD may be due to T. gondii Infection
Metabolic syndrome (MetS) is a chronic inflammatory disease characterized by abdominal obesity, impaired glucose metabolism, dyslipidemia with elevated triglycerides, low high density lipoproteins, and hypertension [451]. Nagahori et al. [452] showed that in their cohort of 107 quiescent IBD patients (76 with UC, 31 with CD; 21.1% of males, 12.9% females) the prevalence of MetS was comparable to that of the general population. Yorulmaz et al. [453] reported frequent occurrence of MetS with increasing age in 177 patients with IBD (62 had CD and 115 UC), especially in UC than in CD (29.5 vs. 17.7%, P < 0.01). MetS was diagnosed in 10.3% of patients with IBD, under 45 yrs of age, and in 55% of patients over 45 yrs of age (P < 0.0001) [452,453].
Intestinal inflammation in IBD may arise primarily from intraepithelial cells dysfunction due to unresolved endoplasmic reticulum (ER) stress caused by the accumulation of misfolded or unfolded proteins within the ER [454-456]. Impairment of proper ER stress resolution in highly secretory Paneth and, to lesser extent goblet cells within the epithelium, can primary lead to intestinal inflammation [454,457,458]. The range of environmental factors possibly leading to ER and development and/or perpetuation of intestinal inflammation include those associated with bacteria, metabolic factors, drug effects, hypoxia, and inflammation itself [455]. It must however be emphasized that congenital or acquired oral T. gondii infection should also be included in these environmental factors because the host cell ERparasitophorus vacuole interaction provides a route of entry for the parasite antigens cross-presentation in T. gondii -infected dendritic cells [15]. Recently, Prandota [210] proposed to term this close relationship a “perpetuum mobile-like machinery” because the parasite or its fragments remain in the host cell permanently, thus being a constant source of various antigens. C-reactive protein has been shown to be an objective marker of intestinal inflammation and fecal lactoferrin important for the innate defense against T. gondii infection (sic!) and calprotectin may be helpful in differentiating patients with IBD from those with functional disorders and to predict clinical relapse [459].
Similarities between MetS in IBD and Clinical/ Metabolic Changes Found in Patients With ASD as well as These in the Host Cell Proteome Caused by T. gondii Infection
Children with autism had a serious prevalence of at-risk-for overweight and overweight [460-463]. Xiong et al. [460] found that among 380 boys and 49 girls with ASD, the prevalence of at-risk-for or being overweight were 31.8% and 17% in 2-5 years old group, and 37.9% and 21.8% in 6-11 years old group. Other authors reported [461] that the prevalence of at-risk-for-overweight was highest in the 12-17.9 years old group, and in a large study of 20,031 Japanese children and adolescence with mental retardation that included 413 children with autism, the prevalence of obesity was 22% in boys and 11% in girls [463]. Proinflammatory cytokines, such as IL-1, TNF-α, sTNFR-1 and sTNFR-2 have been shown to be elevated in obese patients/animals and to decline with weight loss [464,465], in general population [452], as well as in obese prepubertal children [466,467]. In those children statistically significant positive correlations were found between serum leptin and IL-2, IL-1β, IL-6 or TNF-α concentrations [466]. There was also a significant negative correlation between leptin and IL-2 serum concentrations [466]. Wiest et al. [468] showed significantly changed plasma fatty acid profiles in children with autism, including phosphatidylcholine and phosphatidylethanolamine, and suggested that the function of perioxosome and the enzymes of the perioxosome involved with fatty acid metabolism may be affected in this clinical entity.
T. gondii infection induced lipid metabolism alterations in the murine host [469]. A significant decrease in plasma HDL and total cholesterol concentrations was first noted at day 14 and persisted to day 42 after inoculation by oropharyngeal gavage with 8 cysts, and at day 42 serum LDL levels correlated with the brain cysts counts of above 300 (44% of the infected mice), while the change in HDL between days O and 42 correlated with both the overall mean cyst count and cyst counts above 300 [469]. The parasite can synthesize lipids de novo, as well as actively scavenge specific lipids and sterols from the extracellular milieu, lipid bodies, and mitochondria [464,470-473]. It appeared that T. gondii employs host low-density lipoprotein receptor (LDLr) to acquire cholesterol and favor its growth, and in the presence of hypercholesterolemia the parasites are able to acquire cholesterol-rich lipoproteins through an alternative host receptor, and overcome LDLr deficiency, favoring host parasitism and impairing lipid loading of foam cells [465]. It must be also noted that mast cells, like macrophages and T lymphocytes, are inflammatory cells that participate in the pathogenesis of inflammatory diseases such as cardiovascular complications and metabolic disorders, including involvement in insulin resistance and type 2 diabetes, therefore playing an important role in development of MetS [474]. Mast cells are also believed to play an important role in development of ASD [320,321,353].
Studies of Nelson et al. [472] showed that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modification, and revealed a complex and intimate molecular relationship between host and parasite. Modulation of the host metabolism was intrinsic to the cell response to infection, and overall out of 30 affected proteins 16 proteins were downregulated, with only 10 being upregulated and the remaining 4 modulated [472]. Each of the 4 proteins directly involved in lipid and sterol metabolism identified by Nelson et al. [472] (carbonyl reductase 1, vigilin, cargo selection protein TIP47, and LRP protein) was downregulated in response to infection. Also, several proteins involved in glycolysis, amino acid metabolism, and other aspects of intermediary metabolism essential to lipid synthesis exhibited marked changes [472].
Differences in Susceptibility of the Cholinergic and Adrenergic Myenteric Neurons to Damage Caused by T. gondii Infection may be Partly Responsible for Some Distinct Pathophysiological Features Characteristic for CD and UC
Hibi et al. [475] suggested that several environmental factors differently affect clinical course of IBD, with cigarette smoking identified as the strongest agent (Table 43). Interestingly, nonsmoking, appendectomy, and breastfeeding may even exert a beneficial effect on clinical course of UC (a disease largely of nonsmokers and former smokers), while smoking has a detrimental influence on CD. They believed that the potential mechanisms involved in this dual relationship include changes in humoral and cellular immunity, cytokine and oxygen free radicals production [475]. These observations are in agreement with the fact that the control of T. gondii infection in a variety of host cells is mediated through production of various cytokines, including IFN-γ, TNF-α, IL-1β, NO, and reactive oxygen/nitrogen species [476,477]. One cannot therefore exclude that differences in the feces transit time through the small and large intestines, and different numbers of tachyzoites, bradyzoites, and/or oocysts finally reaching these parts of the gastrointestinal tract may be partly responsible for development of some characteristic clinical features of UC and CD.
Table 43: Possible effect of some environmental factors on clinical course of UC and CD [475].
It must be noted that nicotine causes dose-dependent superoxide anion generation by human neutrophils [478], and production of reactive oxygen/nitrogen species is one of six important mechanisms that control T. gondii survival in both phagocytic and non-phagocytic cells [477]. It must be noted that treatment with cholinergic agonists improved behavioral abnormalities in autism [315,316]. Moreover, recently Ghia et al. [479,480] demonstrated the protective, antiinflammatory function of the vagus nerve in murine models of acute and chronic relapsing colitis and a role for nicotinic receptors and macrophages in mediating this function. Beneficial therapeutic effects of smoking observed in patients with UC and summarized by Hibi et al. [475] may therefore be explained by the fact that proinflammatory cytokine levels and excessive inflammation can be regulated by specifically augmenting cholinergic signalling via the efferent vagus nerve and/or applying selective cholinergic modalities targeting the α7 subunit-containing nicotinic acetylcholine receptor [317,318,481]. Furthermore, it seems that because proliferation of T. gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production [482], the enhanced tobacco smoke exposure potentiated superoxide anion generation by human neutrophils [483] appeared to be advantageous for the UC patients infected with the parasite. On the other hand, nicotine was found to inhibit production of proinflammatory mediators in human monocytes [481]. The nicotine action might be at least in part responsible for the detrimental effects of smoking reported in patients with CD (Table 43), whose inflammatory processes probably generate less neutrophils locally and in the systemic circulation than in UC individuals. Moreover, there was a relationship between NO toxicity and cholinergic receptors in the brain of patients with ASD. In the cortex of autistic patients, the cholinergic receptors known to be sensitive to NO toxicity were found to be decreased [314]. (Nb. age-related cell loss in the small and large intestines of rats occur exclusivelly in the cholinergic subpopulation, but it appeared from the somatic hypertrophy and the presence of swollen axons that the nitrergic neurons were not completely spared from the effects of age [94]). ZoroÄŸlu et al. [45] and Sweeten et al. [46] reported elevated plasma nitrite (a metabolite of NO) levels in autistic subjects, and Sogut et al. [22] found increased NO levels in red blood cells of patients with autism. A positive correlation was found between nitrates and IFN-γ concentrations, indicating that elevated plasma NO may be related to IFN-γ activity in ASD [46]. This is not surprising because the induction of iNOS is mediated by some cytokines, namely IFN-γ, TNF-α and IL- β1 [484], and autistic individuals have persistent neuroinflammation [39,41,44]. Finally, it should be noted that IBD is associated with a differential expression of VIP and nNOs neuronal subpopulations whithin the two major enteric plexi, likely due to phenotypic switch [485]. In pediatric patients with CD the sumbucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons, while in the myenteric plexus, there was a significant increase in the percent of NOS neurons. Boyer et al. [485] suggested that these changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease. Nb. development of human enteric nervous system has been characterized by the early (between 9 and 12 weeks’ gestation) appearance of adrenergic and cholinergic nerves, and by 12 weeks’ gestation nitrergic neurons had appeared in the myenteric ganglia and had begun plexus formation [486]. By 23 weeks’ gestation nitrergic innervation has matured and the onset and pace of development of nitrergic innervation are similar to adrenergic and cholinergic innervatiuon and occur before peptidergic innervation, thus NO has a pathophysiological role in development of gut motility disorders [486].
Disturbances in the carbohydrate metabolism due to T. gondii invasion might be at least in part responsible for the reported link between the increased sugar intake and development/worsening of IBD clinical course, especially in Crohn’s disease [475,487] (Table 43). Studies on the modulation of the host cell proteome by the parasite showed that six proteins were involved in carbohydrate metabolism (aldose reductase, aldehyde dehydrogenase 1A3, aldehyde dehydrogenase X, hexoaminidase B, phosphoenoylpyruvate carboxykinase, and 6-phosphogluconolactonase) [488]. The glycolytic pathway also exhibited considerable modification during such infection with six enzymes showing either an increase or modulation (aldolase A and B, enolase, glyceraldehyde 3 phosphate dehydrogenase, phosphoglycerate kinase, and pyruvate kinase) and only one of which showed a decrease in expression (triose phosphate isomerase) [488] Table 44 presented some changes in the proteomes of human foreskin fibroblasts proteins caused by infection with the parasite and proteomic expression of some similar biomarkers reported also in the rat model of IBS due to mothball odor.
Table 44: Selected changes in the proteomes of human foreskin fibroblasts (HFF) caused by T. gondii infection (acc. to Nelson et al. [488]; with own modification), and proteomic expression analysis of colonic mucosa in a rat model of IBS established by a special odor of mothball as a conditional stimulation (Ding et al. [489,490]; with own modification).
In individuals with UC oral contraceptives may enhance intensity of clinical course [475]. Deleterious effects of estrogen and progesterone derivatives contained in contraceptive preparations may be associated with modulation of the innate and acquired immunity of the host resulting in disturbances of immune balance between the immunocompetent host and latent T. gondii infection [477]. Estrogen significantly increased IFN-γ and IL-2 mRNA in concavalin-A activated thymocytes, splenic lymphocytes, and in enriched splenic T cells [493], regulated transcription factors STAT-1 and NF-κB to promote inducible NO synthase and inflammatory responses [494], and dysregulated Tand B-cell balance by inducing selective T-cell hypoactivity and B-cell hyperactivity [495]. Moreover, progesterone-induced blocking factor (PIBF), a molecule with inhibitory effects on cell-mediated immune reactions, by acting on the phospholipase A2 enzymes interfered with arachidonic acid metabolism, induced a type 1 to type 2 cytokine shift by upregulating the production of type 2 cytokines, and controlled NK activity modulated cytokine production by lymphocytes [496-499].
Finally, possible beneficial effects of breastfeeding on clinical course of UC summarized by Hibi & Ogata [475] may be associated with strong anti-T. gondii activities of Lf contained in the breast milk.
T. gondii Transmission in Eukaryotic Cells Acting as a “Trojan Horse”
T. gondii transmission may occur by eating uncooked or undercooked meat, contaminated vegetables, cat feces [500], blood transfusion [501-503], materno-fetal transmission [504] during transplacental passage of blood cells [505], via various solid organs allografts [506], bone marrow transplantation [507], allogeneic stem cell transplantation [508], sputum [509], breast milk [510,511], and semen [512]. T. gondii can invade and multiply inside any nucleated cell type including epithelial cells and blood leukocytes [513,514]. A preference to infect and multiply inside myeloid cells in vitro has been reported [336] and several studies indicate that dendritic cells and monocyte/macrophages function as systemic parasite transporters (Trojan horses) during infection in mice [514-519]. The parasite can be transmitted from infected dendritic cells to NK cells [520]. NK-cells and T-cells have been suggested to contribute to parasite dissemination via a sequestering mechanism [520-522].
Maternal-Fetal Microchimerisms and Transmission of T. gondii
The risk of infection with T. gondii is 0.1% to 1% of all pregnancies [523], and the risk of intrauterine infection of the fetus increases during pregnancy from 10-20% after primary maternal infection in the first trimester to about 59-90% in the third trimester [387,524-526] (Table 37). Tachyzoites can invade and multiply within placental trophoblastic cells [527], and are located directly at the interface between maternal and fetal compartments. Therefore, cytotrophoblasts play a key role in the maternal-fetal transmission of the parasites, especially that the fetus is swallowing the amniotic fluid, up to 1 liter per day (about every 30 min at 24 weeks of gestational age, every 40 min at 32 weeks, and every 80 min at term) [528,529].
The prenatal transfer of nucleated maternal cells into the fetal circulation can occur as early as the 13th week of gestation [530]. This prenatal cell trafficking is a potential mechanism for the mother to affect the development of the immune system of the fetus. In one study, overall 33% (10/30) subjects had at least one source of microchimerism in CD66b+ cells [532]. Maternal microchimerism was found to be more common than fetal microchimerism, 40% vs. 15%, respectively (P = 0.05) and was present at higher levels (P = 0.03) [531]. Studies of Jonsson et al. [532] showed that the maternal cells of lymphoid and myeloid lineages and hematopoietic progenitors were widely distributed in the second-trimester fetuses. It must be noted that the second-trimester histopathological placental findings in maternal-fetal inflammatory response syndrome showed 65.3% acute inflammatory response of the 354 placentas and in 98.7% of the cases, inflammation was classified as maternal inflammatory response and in 49.8% as fetal inflammatory response analyzed [533]. Microchimerism of maternal origin persists well into adult life [534]. This may also provide a possible route for the prenatal transmission of infectious agents, such as the intracellular parasite T. gondii, from the mother to the fetus [530]. It must be emphasized that fetal-maternal and maternal-fetal microchimerisms involve the colonization of different organs and tissues, and therefore may play an important role in T. gondii trafficking as a “Trojan horse” in eukaryotic cells.
Maternal Microchimerism during Physiologic Swallowig of Amniotic Fluid by the Fetus May Cause Development of Serious Gastrointestinal Tract Pathology Before and After Birth
Recently, it was reported that unrecognized ingestion of T. gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America [535]. Moreover, studies showed that the inflammatory bowel syndrome has origins in the childhood socioeconomic environment [536,537].
Physiologic swallowing of amniotic fluid by the fetus may have pivotal pathophysiological consequences because in neonates inflammatory lesions involving esophagus, stomach, and duodenum, as well as gastro-esophageal reflux, are frequent findings with unknown etiology [538-540]. It must be noted that chronic ulcerative colitis [541-544], and Crohn’s disease [545-548] have been reported already in newborn infants. Among patients with UC, 20% are younger than 20 years of age and 1% are infants [549,550]. It was found that the early onset of Crohn’s disease may be at least in part explained by markedly reduced intracellular TH1 IFN-γ levels in peripheral blood of children with this entity compared with controls (P < 0.006) [551]. It must be noted that in mice following peroral infection with T. gondii IFN-γ induced Fas-dependent apoptosis of Peyer’s patch T cells causing a remarkable decrease in the numbers of T cells in the Peyer’s patches of the small intestines [552]. It was also demonstrated that CD11c- and CD11b-expressing mouse leukocytes from the mouse small intestine transported single T. gondii tachyzoites to the brain extravascular space [517]. After intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer’s patches, and the mesenteric lymph nodes were parasitized, while in the blood, parasites were associated with CD11c- CD11b+ monocytes [517]. These findings are in line with our reasoning that T. gondii infection of the fetus may be responsible for early development of the gastrointestinal tract and brain abnormalities because IFN-γ and p47 GTPases, a new family of IFN-γ-induced genes, play a crucial role in the protective immunity against the parasite [477,553,554] and susceptibility of pregnant mice and maternal-fetal transmission of the parasite are type 2-dependent [555,556]. On the other hand, one cannot exclude that virulent T. gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL-12 and IFN-γ and high level apoptosis [557,558], which may be partly responsible for development of NEC in some newborns.
Fetal microchimerism during pregnancy and transmission of T. gondii
The trafficking of fetal cells into the maternal circulation starts very early during pregnancy at approximately 4th to 6th week of gestation [559,560]. A greater number of fetal cells transfer into the maternal circulation than do maternal cells into fetal circulation [505,561]. This traffic of cells is primarily composed by immune cells (T and B-lymphocytes, monocytes, NK cells), including hematopoietic stem cells CD34+ and CD34+/38+ committed to early B and T-cells with the capacity for multilineage differentiation [562]. The number of fetal progenitor cells circulating in the blood of a pregnant woman, has been estimated to be 0-2 per ml, but it can vary according to the gestational age. In normal second-trimester pregnancies, the number of fetal cells in the maternal circulation was found to be 1-6 cell/ml of maternal venous blood. After delivery, this fraction rapidly decreases, and 30- 50% of healthy women have fetal cells in their blood from four weeks to decades postpartum [563]. Pregnancy-associated progenitor cells can survive in the maternal bone marrow representing a long-term reservoir of stem cells [564,565]. The exchange of fetal microchimeric cells between non-HLA identical twins also is a common event [566] and approximately 8% of human twin pregnancies and 21% of triplets are chimeric in their blood cell populations from their siblings [567]. Table 45 presented fetal-maternal cell trafficking involving a broad range of different cell types, including maternal and fetal cells with stem/progenitor phenotype [568,571,572]. Furthermore, trisomy 21 was reported to result in especially high numbers of maternal cells in the infant [573], which may support recent suggestion that T. gondii infection is responsible for development of Down syndrome [574].
Table 45: Different types of cells involved in fetal-maternal trafficking (acc. to Klonisch et al. [568]; with own modification).
Possible link between bidirectional transmission of T. gondii and/or its antigens in fetal and maternal cells and development of primary biliary cirrhosis and several other autoimmune diseases
The origins of autoimmunity are still elusive despite marked advances in immunology [575]. Because T. gondii can be transmitted bidirectionally in fetal and/or maternal cells, one cannot exclude that the majority of individuals in the group of patients with primary biliary cirrhosis studied by Fanning et al. [576] suffered from the parasite infection. The extracellular mtDNA found by Zhang et al. [577] in serum of autistic children may eventually represent DNA of the parasite since Bidgoli et al. [578] detected T. gondii DNA by PCR analysis in a vitreous sample in a 13-year-old immunocompetent patient with toxoplasmic chorioretinitis even with repeated negative serology for T. gondii. Possible important role of transplacentally-acquired antibodies to infectious agents as the cause of neuromental disorders proposed by Nahmias et al. [579], also may support this reasoning. Moreover, the markedly increased female to male ratio found in these diseases is in line with our recent finding that females are more susceptible to the parasite infection than males [444].
It is interesting that anti-mitochondrial antibodies directed against various proteins of the outer and inner mitochondrial membrane (AMA-M2) have been clinically detected also in patients with biliary cirrhosis [580]. Moreover, it was found that fetal microchimeric cells are present in high numbers in women with several autoimmune diseases, including primary biliary cirrhosis (Table 46). This is not surprising because Khosrotehrani et al. [562] found that fetal cells have multilineage potential to maternal tissue, and several authors [593,594] provided evidence of maternal microchimerism underlying pathogenesis of biliary atresia (2 X-chromosome and maternal chimeric CD8+ T cells, respectively). It must be noted that no fetal cell microchimerism was demonstrated in the patients with primary biliary cirrhosis [595,596]. These findings are therefore in agreement with our suggestion that maternal and/or fetal cells may serve as a Trojan horse for T. gondii dissemination in eukaryotic cells, especially that high levels of IgG antibodies against T. gondii were found in the sera of patients with autoimmune thyroid diseases (Hashimoto’s thyroiditis and Graves’ disease) compared with controls (56.5% vs. 38.0%, P < 0.02). Another study performed also in nonautoimmune individuals found that infection with the parasite was associated with a high autoantibody burden.
Table 46: Diseases associated with fetal and/or maternal microchimerism (acc. to Klonisch et al. [568]; with own modification).
Amelioration of Experimental Colitis was Found to be Associated with Histone Hyperacetylation. Toxoplasma Gondii Expresses a Histone Deacetylase Class I Enzyme Homologous to Human HDAC3, and Valproic Acid, an Antiepileptic Drug and Histone Deacetylase Inhibitor, has Potent Antitoxoplasmal Activity
Recently, Glauben et al. [597] demonstrated that inhibitors of histone deacetylases (HDAC) exerted strong protective effects in various models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines. This is an important finding because in Crohn’s disease and ulcerative colitis, disease severity can be ameliorated by TH1 inhibitory strategies, such as infliximab (the neutralizing anti-TNF-α Ab) [598], while for ulcerative colitis, anti-TH2 means, such as IL-13-neutralizing compounds, are efficacious [599].
HDAC comprise of a family of enzymes that participate in the regulation of chromatin structure, gene expression, and cell signalling in eukaryotes [600]. One of the most important mechanisms in chromatin remodelling is the posttranslational modification of the NH2-terminal tails of histones by acetylation, which contributes to a “histone code” determining the activity of target genes [601]. Acetylation of histone proteins neutralizes the positive charge on lysine residues and disrupts nucleosome structure, allowing unfolding of the associated DNA, subsequent access by transcription factors (e.g. Mad-1, BCL-6, ETO), and changes in gene expression [602]. Acetylation of core nucleosomal histones is regulated by the opposing activities of histone acetyltransferase and deacetyltransferase HDACs [603]. HDACs catalyze the removal of acetyl groups on the NH2- terminal lysine residues of core nucleosomal histones, and this activity is generally associated with transcriptional repression [599]. Histone deacetylation and DNA methylation are two major epigenetic modifications that contribute to the stability of gene expression states. Perturbing DNA methylation or disrupting the downstream response to histone deacetylases by genetic or pharmacologic means has revealed a critical requirement for epigenetic regulation in brain development, learning, and mature nervous system stability [604].
HDAC inhibitors, such as valproic acid (VPA), a short-chained fatty acid, increase the accumulation of hyperacetylated histones H3 and H4, directly affecting chromatin structure, and thereby, the relationship of the nucleosome and the gene promoter elements [600,605,606]. Inhibition of HDAC in vitro was associated with a significant dosedependent suppression of proinflammatory cytokines, stimulation of apoptosis, and a local increase in histone acetylation [597,607].
VPA is an effective inhibitor of HDAC with an IC50 (0.4 mM) well within the therapeutic range of VPA (0.35-0.7 mM in serum). T. gondii expresses a HDAC class I enzyme homologous to human hdac3, and VPA inhibited the parasite tachyzoite proliferation at concentrations only a few times greater than its respective IC50 [607]. VPA resulted in amelioration of disease in dextran sodium sulfate-induced colitis (a marked reduction in weight loss and histologic signs of the inflammation) associated with suppression of IFN- γ, IL-6, IL-1β, and MIP-2 [600]. In parallel to the beneficial effect observed, a dosedependent increase in histone 3 acetylation at the site of inflammation was observed under VPA treatment [600]. Also apicidin’s (a novel antiprotozoal cyclic tetrapeptide agent) antiparasitic activity appeared to be due to low nanomolar inhibition of histone deacetylase, a key nuclear enzyme involved in transcriptional control, which induces hyperacetylation of histones in treated parasites [608]. Because VPA and its sodium salt inhibited in vitro replication of T. gondii tachyzoites (Table 42), one cannot exclude that the preparations may be effective also in amelioration of some gastrointestinal signs and symptomes observed in anti-T. gondii seropositive patients with ASD and/or other neurodegenerative clinical disorders [574,609], including Alzheimer disease [610]. This reasoning may be supported by the finding that VPA was found to be neuroprotective in experimental cerebral ischemia and the protection mechanisms may involve HDAC inhibition and heat shock protein 70 time-dependent induction [611].
Colonization of the Pouches Formed for Ulcerative Colitis by Sulfate-Reducing Bacteria May Be Associated With a Defense Reaction of the Intestine against T. gondii Infection
Duffy et al. [612] isolated sulfate-reducing bacteria from ulcerative colitis pouches in 8 of their 10 patients with UC. It was found that these bacteria were exclusive to patients with the background of UC because the levels of Lactobacilli, Bifidobacterium, Bacteroides sp, Clostridium perfringens, enterococci, and coliforms were similar in both pouch groups, i.e. UC and familial adenomatous polyposis [612]. The authors suggested that since sulfate-reducing bacteria are specific to UC pouches, they play a role in the pathogenesis of pouchitis. Further studies of Ohge et al. [613] showed that in active pouchitis, sulfate-reducing bacterial counts were found to be significantly higher than in those who never experienced pouchitis. It was reported that pouch contents of the patients with ileoanal pouches and ongoing pouchitis or an episode within the previous year, released significantly more hydrogen sulfide (a highly toxic gas produced by certain fecal bacteria, which causes tissue injury in experimental animals) than did the contents of patients who never had an attack of pouchitis and those with longstanding inactive disease [613]. In addition, pouch contents from familial adenomatous polyposis patients produced markedly less hydrogen sulfide than did any group of nonantibiotic treated UC patients [613]. These findings may suggest that the colonization of pouches formed for ulcerative colitis by sulfate-reducing bacteria is associated with a defense reaction of the intestine against T. gondii infection. This motion may be supported by the fact that electric charges on cell surface play an important role in some cellular processes, including cell-cell interaction, cellular differentiation and endocytosis [614-616]. It was demonstrated that the parasite invaded nearly all types of mammalian cells and it seems that recognition of cell surface sulfated proteoglycans may contribute to such invasion [617]. Cell surface heparan sulfate (HS) and glycans, containing sialic acid have been shown to act as potential receptors for the parasite [617-619]. HS consists of alternating units of N-acetyl glucosamine and glucuronic acid or iduronic acid variously decorated with sulfate residues. The chains are covalently linked to proteoglycan core proteins on the surface of cells and in the extracellular matrix [620,621]. Sialic acids are found on the termini of the glycan chains on glycoproteins or glycolipids. Loss of HS chains or sialic acid from cellular glycoconjugates results in significant reduction of T. gondii infection in vitro [618]. T. gondii recognizes host cell sulfated proteoglycans by examining a wide range of human cell types, and ligands on the parasite are capable of recognizing both highly sulfated glycans like heparin, consisting of glucuronic or iduronic acid and sulfated glucosamine, and the relatively less sulfated chondroitins, consisting of glucuronic acid and variably sulfated N-acetylgalactosamine [617]. Jacquet et al. [622] demonstrated that the parasite surface antigen SAG3 mediates the attachment of T. gondii to cell surface proteoglycans, and it appeared that proteoglycan sulfation was critical for SAG3 adherence to target cells HS proteoglycans. These findings apply also to other microorganisms migration and invasion of host cells, such as Plasmodium sporozoites [623], human cytomegalovirus [624], Listeria monocytogenes [625], and herpes simplex virus [626]. Thus, one cannot exclude that the sulfatereducing bacteria exclusive to patients with ulcerative colitis pouches plays an important role in markedly diminishing attachment of T. gondii to the host intestinal cells that results in further dissemination, invasion and proliferation of the parasite inside the host intestinal cells. Moreover, the parasite may, at least in part, be responsible for development of ileal and colonic pouches in patients with UC and Crohn’s disease because it causes GI tract inflammation associated with enteric glial cells dysfunction/loss and local damage of myenteric neurones in the intestinal wall [107,150,151,391,627]. Development of acute and chronic diverticular disease also may be caused by post inflammatory damage to the ENS [628] caused by T. gondii infection because of marked changes in the intestinal wall morphology reported in experimental animals (Tables 9-13) affecting its integrity.
Development of Celiac Disease May Be Associated With T. gondii Infection
Several studies showed a strong association between celiac disease and various unexplained “soft” neurologic and psychiatric manifestations, such as headache, migraine (in one study tension type headache and migraine had about 40% of children with celiac disease), visual disturbances, cerebral calcifications, developmental delay, hypotonia, learning disorders, cognitive impairment, and attention deficit/hyperactivity disorder, as well as chronic “hard” disorders, such as epilepsy, chorea, chronic neuropathies, cerebellar ataxia, myoclonic ataxia, dementia, depression, and progressive leukoencephalopathy [629-636]. It was believed that neurologic complications of celiac disease could be a nonspecific result of chronic poor nutritional condition or caused by specific nutritional deficiences, such as lowered levels of vitamin B12, E, D, folic acid, pyridoxine, or carnitine [629,633]. At present, however, celiac disease is diagnosed earlier and severe malabsorption is rare, and therefore, it has been suggested that these disorders may result from the fact that the brain is particularly vulnerable to prolonged exposure to gluten with its multisystem immunologic and inflammatory effects [629,633,635].
Celiac disease is characterized by mixed cellular infiltrate of jejunal mucosa by B and T-cell lymphocytes and macrophages or dendritic cells. Gliadin-activated T cells are suggested to play an important role including production proinflammatory cytokines (including IFN-γ and IL-6), increase of cell infiltration and their activation [637,638]. The patients with active celiac disease showed to have higher percentage of samples expressing IL-1β, TNF-α, TNF-β, IL-2, IFN-γ, TGF-β, IL- 10 mRNA compared with controls [639]. The expression of both TH1 (IFN-γ, IL-2) and TH2 (IL-4, IL-10)-associated cytokine transcripts in the same biopsies and peripheral blood cells of these subjects was implying activation of TH0 cells [639]. Hansson et al. [637] found, however, that peripheral blood cytokine-producing cells of children with celiac disease secrete cytokines compatible with a type TH1 response. Increased levels of serum NO have also been reported in such patients, and such sustained high-output NO production occurring in chronic inflammation, may be deleterious to the tissues [638,640]. In addition, food antigens, gluten, gliadin, and their proteolytic fragments activated macrophages (producer of reactive nitrogen oxide radicals) and significantly enhanced TNF-α, IFN-γ, IL-6, NO and iNOS mRNA production [637,638].
MRI revealed unilateral and bilateral white matter lesions of different degrees of intensity, varying between smaller spot and larger flat lesions in one study of 15 children (20%) with neurologic manifestations and biopsy-proven celiac disease (mean age of 11.6 years, 10 girls, 5 boys) [629]. These lesions were hypointense in T2 and fluid attenuated inversion recovery sequences and showed biparietal and left-sided predominance (including periventricular hypodensity) [629]. It was believed that they may be ischemic in origin as a result of vasculitis or caused by inflammatory demyelinetion [629]. In the patients with established celiac disease, epilepsy (with an incidence of 1% to 6%) often associated with bilateral corticosubcortical occipital calcifications, was the most frequent neurologic complication [641-645]. It was suggested that in patients who suffer from celiac disease with migraineous or nonspecific headache, malabsorption did not play a significant role in the pathogenesis of this symptom and inflammatory processes and one should look for other causes, including immunologic mechanisms [633]. Thus, basing on these facts and reasoning one may suggest that all the above-mentioned immune state irregularities of the patients with neurologic manifestations associated with celiac disease could interfere with the immune state of the host and T. gondii in the brain finally causing reactivation of latent cerebral toxoplasmosis. The rapid and concentration-dependent changes in CYP-450 enzyme activities in isolated perfused rat livers due to administration of exogenous NO donors [646], may at least in part, support this suggestion.
The MRI findings in the brain of those subjects, including cerebral calcifications considered as vascular calcified malformations [641], may therefore be a result of chronic subclinical cerebral vasculitis due to T. gondii infection/inflammation. The improvement of neurologic symptoms in some patients after introduction of gluten free diet [629,632,633,641,645] (in few children, even hypodense areas in the white matter around calcifications decreased or disappeared after such a diet [631]) may be explained by amelioration of immune irregularities caused by this triggering factor and therefore diminished pathologic interference in the immune state of the host and the parasite in the brain. The above-presented reasoning may be supported by the opinion of Nejad et al. [647] that celiac disease increases the risk of T. gondii infection in pregnant women. However, this conception may also be alternatively interpreted that the parasite plays an important role in development of celiac disease. Although Plot et al. [359] reported that the rate of positive anti-T. gondii IgG antibodies in their cohort of 90 celiac patients and 297 healthy individuals was similar (23.3% vs. 25.9%, P = 0.36), genetic predisposition should be taken into consideration, because the celiac patients also had markedly increased antibodies against other infectious agents, antirubella IgG (P < 0.05), anti-CMV IgG (P < 0.01), and anti-EBV capsid antigen IgG (P < 0.01).
Finally, it was reported that some children with celiac disease suffer with eosinophilic esophagitis concomitantly, and the coexistance of both these clinical entities is more frequent than anticipated [648- 650]. It was believed that based on differences in pathophysiology and affected gastrointestinal compartments, a causal relationship between these two clinical entities seems unlikely [650], but one cannot exclude that development of eosinophilic esophagitis in children with celiac disease may be associated with T. gondii infection.
Clear Cell Colitis with Presence of Foamy Macrophages May Be Caused by Oral T. gondii Infection
Presence of characteristic foamy macrophages observed in histopathological hematoxylin and eosin-stained specimens is a crucial diagnostic factor in foamy colitis, excludes Crohn’s disease and UC, and confirms mild and moderate course of that type of inflammation in children [651-654]. The induction of foamy macrophages packed with lipid bodies have been reported in several clinical pathologic states associated with chronic proinflammatory stimuli, including T. gondii [465] and human Mycobacterium tuberculosis infections [655].
Foamy colitis (microscopic colitis) [651,654] may be caused by oral prenatal and/or postnatal infection with T. gondii. It was reported that bradyzoites are resistant to gastric digestion and remain orally infective whereas tachyzoites are destroyed by gastric juice within 1 hr [656-658]. However, Dubey [659] showed that tachyzoites occasionally survived at acid peptic digestion for 2 hrs, and the strain of T. gondii did not affect the susceptibility of tachyzoites to acid pepsin solution. In orally inoculated animals extracellular tachyzoites were infective, although the infectivity was dose-dependent [659]. One cannot therefore exclude oral infection with T. gondii in breastfed infants of infected mothers because it is known that the parasite is disseminated in breast milk [507,508], and postnatally gastric acid secretion displays a biphasic pattern with the lowest gastric acid concentrations between 10 and 30 days of life approaching the lower limit of adult values by 3 months of age [660]. The parasite is disseminating in the body as a Trojan horse in various eukaryotic cells, including macrophages, and division rate of intracellular unprimed T. gondii tachyzoites in alveolar, peritoneal or monocyte-derived macrophages is rapid [335]. Successful intracellular replication of tachyzoites and a substantial increase in membrane mass of PV is dependent on diversion and use of lipid resources from its host, especially long-chain fatty acids and cholesterol [468]. T. gondii employs host low-density lipoproteins (LDL) receptor to acquire cholesterol [465] and diverts it for cholesteryl ester synthesis and storage in lipid bodies [468,661,662], leading to formation of foam cells [465]. Macrophages convert into foam cells through a dysregulation in the balance between the influx and efflux of LDL particles (containing cholesterol, triacylglycerides and phospholipids) from the serum. It must be emphasized that foamy macrophages are not only the product of an inflammatory response but amplify that response through production of PGE2 and leukotrienes [663,664], and appear to be a key player in both sustaining persistent bacteria and contributing to development of human tuberculosis granuloma and cavitation [655]. Foam macrophages characteristic for microscopic colitis probably play a similar role.
Enlarged Mesenteric Lymph Nodes Frequnetly Seen at Imaging in Patients with IBD, Children with Abdominal Pain, and in Asymptomatic Children may be Associated with T. gondii Infection
Mesenteric lymphadenopathy is commonly found in patients with IBD, both in CD and UC [665,666], although it is more frequently observed in CD [665,667]. The lymph nodes described as large or prominent may be seen at the mesenteric root, the mesenteric periphery, or the right lower quadrant. Inflammatory changes in the small or large bowel are usually but not always present [667].
Enlarged mesenteric lymph nodes (MLNs) have been frequently reported in children with acute or chronic abdominal pain during abdominal CT or ultrasonographic examination in children with mesenteric lymphadenitis [668-671]. These abnormalities may be found also in asymptomatic children [671-674]. In one study [671], enlarged MLN were detected in 55 of 189 asymptomatic children (29.1%) aged 2 to 10 years and the longitudinal diameter of the lymph nodes ranged between 5 mm and 19 mm. These lymph nodes were arranged in clusters (3 to 9 in number in a cluster) and some children had associated minimal mural thickening of the terminal ileum. It was suggested that this is a non-specific and usually non-pathological finding with the most common location around the iliac vessels [670,671]. Watanabe et al [674] also detected enlarged ileocecal lymph nodes in asymptomatic children examined by ultrasound with similar incidence (26 of the 112 cases, 21%). In patients with mesenteric lymphadenopathy, the clinical presentation is non-specific including abdominal pain, fever and leukocytosis mimicking a broad spectrum of different clinical diagnoses, such as acute appendicitis, infectious enterocolitis and pyelonephritis [668,671,675].
It must be noted however that the above mentioned enlargement of MLNs in young and adult patients and healthy individuals may be caused by toxoplasmosis because: a) toxoplasmosis affects about a third of gobal human population, b) in experimental animals oral T. gondii genotype III or II inoculation with oocysts (sporozoites)/tachyzoites/ tissue cysts (bradyzoites) also frequently resulted in an increased number of neurons per ganglion and hypertrophy of myenteric neurons [204,205,207,208,211,212,270], and c) oral infection with the parasite spreads locally to MLNs and distant organs by invasion of lymphatics and blood [676].
Human Milk Lactoferrin (Lf) May Be, At Least In Part, Responsible For the Delayed Onset of ASD in Newborns and Young Infants Because It Plays an Important Role in Host Defense against T. gondii Infection and Dissemination
Important role of breastfeeding and Lf in human milk
Vertical transmission of toxoplasmosis from a chronically infected immunocompetent woman developed probably after maternal reinfection or reactivation of the preexisting disease [677]. Acute toxoplasmosis can be transmitted via breast feeding [678].
Breastfeeding had a protective effect on the incidence of childhood illnesses in the 1st 2 years of life, particularly gastrointestinal disease. Infants who had been partially or fully breastfed initially had significantly lower rates of gastrointestinal diseases at 14-26 weeks, 27- 39 weeks, and 40-52 weeks compared to bottle-fed infants and a lower rate of hospital admission [679]. Breastfeeding during the first 13-26 weeks of life conferred subsequent protection against gastrointestinal illness that persisted beyond the period of breastfeeding itself [679,680]. Exclusive breastfeeding for the first 6 months is recommended by the World Health Organization and considered allergy preventive [681]. Saarinen & Kajosaari [682] found that prevalence of eczema at ages 1 and 3 years was lowest in the prolonged (> 6 months) breastfeeding group of infants compared with intermediate (1-6 months), and short or no (< 1 month) breastfeeding. It was reported, however, that prolonged strictly exclusive breastfeeding for more than 9 months was associated with atopic dermatitis (P < 0.002) and symptoms of food hypersensitivity (P = 0.01) in children with a family history of allergy [681].
Human breast milk has been shown to have antiinflammatory properties and have an immediate and localized salutary action on the gastrointestinal and respiratory tracts of the infant [683]. Human milk, colostrum and bovine milk contain a factor, in part resembling TGF-β activity, which inhibits production of IL-2 [684,685].
Studies performed with milk collected from women 20 to 35 years of age during the first year of lactation showed that the concentrations of Lf, total IgA, and leukocytes fell during the first several weeks of lactation [686]. Afterwards, the levels of Lf and IgA stabilized. Approximately 90% of total IgA in human milk during the first year of lactation was secretory IgA. The concentrations of lysozyme, after falling to a nadir of 20 to 30 μg/ml at 2 to 4 weeks, rose to 200 to 300 μg/ ml by six months and remained elevated [685].
Lf is found in large amounts in most secretions, particularly in milk where its concentration may vary from 1-3 g/L (mature milk) to 5-7 g/L (colostrum) [686-688], and in secondary granules of neutrophils [689]. Plasma concentration of Lf may range from 0.4 to 2 mg/L under normal conditions, but increases to up 200 mg/L in septicemia [690]. Pacora et al. [691] found that Lf was also detectable in 85.4% (229/268) of amniotic fluid samples obtained during transabdominal amniocentesis, not detectable in all fluid obtained in the mid trimester, and detectable in all maternal and cord plasma samples. With advancing gestational age, the concentration of Lf was markedly increasing (r = 0.68, P < 0.0001), and intra-amniotic infection was associated with significant increases in amniotic fluid Lf concentrations in patients with preterm labor [691].
Lf has bacteriostatic and bactericidal activity and plays an important role in the first line of defense against microbial infections, since many pathogens tend to enter the body via the mucosa. Lf can bind with high affinity to a 105 kD receptor on cell membrane, but binding to low affinity binding sites, such as glycosaminoglycans does also occur. The positively charged N-terminus of Lf is responsible for the binding to glycosaminoglycans, such as heparan sulfate, chondroitin sulphate [692]. In addition, the LDL remnant receptor [693] and the 45 kD subunit of asialoglycoprotein receptor [694,695] have been demonstrated to act as receptors for Lf [696]. Binding of Lf to LPS of Gram-negative bacteria prevents priming of neutrophils, leading to an inhibition of ROS production [697].
Spontaneous integrin expression on CD4+, CD8+ and CD19+ lymphocytes at 6 months was significantly lower in breastfed than formula-fed infants (P < 0.05) [698]. Before MMR vaccination, lymphocytes of breastfed children had lower levels of blast transformation without antigen (P < 0.001), with tetanus toxoid (P < 0.02) or Candida (P < 0.04), and lower IFN-γ production (P < 0.03). Fourteen days after the live viral vaccination, only the breastfed children had increased production of IFN-γ (P < 0.02) and increased percentages of CD56+ and CD8+ cells. These findings are consistent with a TH1 type response by breastfed children, not evident in formulafed children [698], important also for host defense against T. gondii infection.
Beneficial immunomodulatory properties and effects of Lf
Lf is an 80-kD, non-hem iron-binding multifunctional LPSchelating glycoprotein of the transferrin family, present at the mucosal surface where it functions as a prominent component of the first line host defense against infection and inflammation [687,699]. Lf is also abundant component of the specific granules of neutrophils and can be released into the serum upon neutrophil degranulation. The protein displays a broad antimicrobial spectrum against Gram positive and Gram negative bacteria, fungi and several viruses, because it is an important component of the non-specific immune system. Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells [700]. At the cellular level, the glycoprotein modulates the migration, maturation and function of immune cells [701]. It interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by CD14-LPS complex, thus modifying activation of endothelial cells [702]. Lf administered orally improved gastrointestinal morphology in growing calves because it enhanced size of Peyer’s patches in the ileum and decreased villous sizes in the jejunum [304]. In addition, Lf increased expression and production of several proinflammatory cytokines, such as IL-1β, TNF-, IL-6, IL-8, NO, granulocyte-macrophage colony-stimulating factor [703-706], number and activity of NK cells [707-709], and phagocytosis-enhancing effect [710]. Moreover, up-regulation of antiinflammatory IL-4 and IL-10 cytokines was found after oral Lf administration in rats with colitis [711]. Lf increased also serum IgG levels and the number of peripheral blood leukocytes, and mRNA levels of various cytokines, such as IL-1β, IL-8, IL10, and IFN-γ in those cells, in response to Lf treatment, were enhanced [304]. Haversen et al. [712] found that in monocytic cells Lf down-regulated the LPS-induced cytokine production (expression of TNF-α, IL-6, IL-8 mRNAs, and secretion of IL-10), and the inhibitory mechanism was suggested to involve the interference of Lf with NF- κB activation. It appeared that Lf was internalized into THP-1 and Mono Mac 6 monocytic cell lines and detected in the nucleoli [712]. Additionally, the mRNA expression of proinflammatory cytokines IL 1β and IFN-γ in blood decreased over 10-week treatment with Lf [304]. It was suggested that immunomodulatory effects of Lf are due, in part, to LPS binding [714] (Table 7). Moreover, orally administered Lf restored humoral immune response in immunocompromised mice [714]. Thus, the immune regulatory properties of Lf may exert beneficial effects in the patients with inflammatory processes of the gastrointestinal tract caused by various microbial pathogens, including T. gondii.
Beneficial effects of Lf in inflammatory diseases
Lf is a potent molecule in the treatment of inflammatory diseases and its major activity is related to the scavenging of free iron, which accumulates in inflamed tissues and catalyzing the production of tissuetoxic hydroxyl radicals. Ambruso et al. [715] found that Lf enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system.
Free radical is thought to play a significant role in the pathogenesis of several disease processes in low birth weight preterm infants including NEC [716], partly because iron is a known catalyst in free radical-mediated oxidation reactions. Raghuveer et al. [716] found that before adding medicinal iron to formula or human milk, significantly more ascorbate and alpha-hydroxyethyl radical production and more lipid peroxidation products (i.e. thiobarbituric acid reactive substances, malondialdehyde, and halothane) were detected in formula. After addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. The authors believed that the presence of greater concentration of iron and the absence of Lf in formula compared with human milk resulted in greater in vitro generation of free radicals and lipid peroxidation products [716].
Davidsson et al. [717] found that fractional iron absorption was markedly lower from breast milk (11.8%; range 3.4-37.4%) than from Lf free breast milk (19.8%; 8.4-72.8%) (P < 0.05). It must be added that iron absorption was higher from human milk than from infant formula or bovine milk [718]. Human fetal small intestinal brush-border membrane receptor for Lf may be responsible for high iron absorption from human milk. The molecular weight of the receptor was 110 kD under nonreducing conditions and 37 kD after reducing circumstances. The binding was pH dependent, with an optimum between pH 6.5 and 7.5. Little binding of bovine Lf or human transferrin to human brushborder membrane vesicles was found [718].
In neurodegenerative diseases where iron deposits contribute to oxidative stress and neuronal death, an upregulated by TNF-α synthesis of Lf by activated microglia in human substantia nigra [719], together with transcytosis of plasma Lf through the blood-brain barrier to limit inflammation processes in the brain, were reported [720]. Lf was found to display antiviral activity against both DNA- and RNA-viruses, including herpes simplex type 1 and 2 viruses (α-herpes virus family), by preventing entry of virus to the host cell, either by blocking cellular receptors, or by direct binding to the virus particles [696]. It appeared that lactoferricin (Lfcin), a residue of 24 aminoacids derived from the N-terminal region of the N-lobe of Lf, also displayed antiviral activity against HSV, but the native protein (Lf) was more potent [721].
Important role of Lf in IBD
During the course of IBD activated leukocytes infiltrate the intestinal mucosa and are detected in feces as they shed into the intestinal lumen. Lf can reflect the activity of neutrophil leukocytes and levels of this iron-binding glycoprotein have been shown to increase dramatically in body fluids during the course of inflammation [722]. Several authors reported that the levels of fecal Lf were markedly higher in IBD patients, especially those with Crohn’s disease, than in controls, patients with bacterial infectious bowel disease, or IBS individuals [723-725]. The levels of fecal Lf were markedly higher in the patients with inactive IBD than in individuals with IBS [725]. Therefore, fecal Lf has been used as a specific and sensitive noninvasive biomarker of intestinal inflammation activity with cut-off values between 60 g/g [723] and 240 μg/g for active Crohn’s disease and 324 μg/g for active ulcerative colitis [722,724].
Lactoferrin inhibits replication of T. gondii in enterocytes
The oral route is the natural portal of entry for T. gondii. Enterocytes are the first cells to be invaded by the parasite when ingested pathogens are released from cysts or oocysts within the gastrointestinal tract [726]. The intestinal epithelium constitutes a unique lymphoid compartment of the gut mucosa immune system, with the presence of intraepithelial lymphocytes that have the ability to lyse T. gondiiinfected enterocytes and to synthesize IFN-γ in infected mice [727]. The capacity of enterocytes to inhibit T. gondii replication after IFN-γ activation through an iron-dependent mechanism may have important implications in the defense against the parasite and its dissemination.
Ingested organisms are released from cysts or oocysts within the gastrointestinal tract and initially invade the intestinal epithelium before disseminating throughout the body. The intestinal epithelium and the underlying mucosal tissues are heavily populated with cells of the local immune system that can make rapid contact with the parasite during intestinal penetration [728]. Intestinal cells have a rapid cell turnover, with a mean cell duration time of 2-3 days in vivo and a few hours in vitro [729]. In vivo, the most prominent expression of secretory component was such in crypt cells [730] and diminished during the maturation of the enterocytes.
T. gondii invaded and proliferated also in cultured primary rat enterocytes. It was found that primary rat enterocytes possess a microbiostatic capacity [728]. Activation of the enterocytes with rat recombinant IFN-γ inhibited T. gondii replication through an irondependent mechanism [728]. Iron diminished IFN-γ-induced activity against T. gondii, and the replication of the parasite was dependent upon the intracellular iron pool. Iron is acquired by enterocytes by a carrier-mediated process, essentially through a transferrin-free form into vesicles [731].
Milk carbohydrate components are interfering with microbial attachment to mucosal membranes
Bout et al. [726] showed that the transcytotic pathway of IgA could interfere with intracellular replication of T. gondii. Otherwise, it was demonstrated that incubation of sodium nitroprusside (SNP), a donor of NO molecules, with Caco-2BBe intestinal epithelial monolayers resulted in time- and concentration-dependent decreases in transepithelial resistance, and widened tight junctions in electron microscopy [23]. Also NO reduced cellular ATP levels and reversibly increased permeability of tight junctions in cultured Caco-2Bbe cells [23]. Unno et al. [24] found that incubation of cultured human intestinal epithelial monolayers Caco-2BBe cells with IFN-γ resulted in upregulation of NO biosynthesis and a marked increase in permeability of intestinal epithelial monolayers.
Lf is relatively resistant to degradation by trypsin and chymotrypsin, and stools of breast-fed babies contain considerable amounts of this glycoprotein [732]. To be able to infect host via the mucosal membranes, where most infections occur, the microbes must attach to the mucosal cells. The process is specific, with different microbes binding to different carbohydrate structures on the cell surface [732]. The carbohydrate components of human milk function as receptor analogues and can therefore prevent microbes from binding to the carbohydrate moiety on the mucosal epithelium to which they are specifically adapted to bind. A similar capacity to prevent microbial attachment to specific mucosal carbohydrate structures has been demonstrated for glycoconjugates in human milk. The milk glycolipid Gb3 prevents binding of Shigella dysenteriae and Shiga-like toxin from enterohaemorrhagic E. coli [733].
Sisk et al. [734] found that gestational age was the only perinatal factor associated with risk of NEC. It appeared that enteral feeding containing at least 50% human milk in the first 14 days of life was associated with a sixfold decrease in the odds of NEC. Prophylactic therapy with recombinant human Lf and the probiotic, Lactobacillus GG (LGG), acted to enhance defenses against invasive E. coli in the neonatal small intestine [735]. It was suggested that rhLf and LGG were therapeutic agents that may reduce NEC and gut-related sepsis in preterm human infants [735]. Sherman et al. [736] hypothesized that Lf helped terminate bacterial invasion of enterocytes via a detachmentinduced apoptosis called anoikis. Death of infected epithelia by anoikis prevents local spread of bacterial pathogens because the bacteria are trapped within the cell. Such infected, apoptotic and sloughed epithelia also cannot infect the lower gastrointestinal tract, and epithelia exit the body with the stool. When neonatal rats were pre-treated with intragastric recombinant human Lf, epithelia with anoikis were found in ileal fluid after enteric infection. They believed that Lf might prevent NE in preterm infants who cannot take human milk [736].
Milk Lf exerts a protective effect against murine gastrointestinal T. gondii infection
In experimental murine toxoplasmosis, all mice orally administered 5 mg Lfcin and challenged with cysts of T. gondii at a dose of LD90 survived 35 days postchallenge [711]. Intraperitoneal administration of 0.1 mg of Lf also prevented death in 100% of treated mice challenged with T. gondii cysts, while 80% of untreated mice died of acute toxoplasmosis within 14 days postchallenge [737].
The sporozoites of T. gondii preincubated with bovine Lfcin showed decreased activity in penetration of mouse embryonal cells [738]. Mice inoculated with 105 sporozoites preincubated with Lfcin showed a higher survival rate than those inoculated with the same number of untreated sporozoites. Rabbits inoculated with 105 sporozoites preincubated with Lfcin shed fewer oocysts than these inoculated with the same number of untreated sporozoites [738]. The mechanism of anti-T. gondii activity induced by Lf was associated with Lf-induced tyrosine phosphorylation in macrophages [739]. Tanaka et al. [739] found that bovine Lf induced tyrosine phosphorylation of a 30 kDa protein in murine macrophages, which may be associated with inhibition of multiplication of T. gondii in the host cells. The density of the specific band of this protein increased dose-dependently and the highest density appeared in the case of mouse peritoneal macrophages incubated with 1,000 μg/mL Lf [739].
Dzitko et al. [740] found that multiplication of the parasite was inhibited by human Lf in human CaCo-2 epithelial cells and in mouse L929 fibroblasts. The intracellular growth of T. gondii was not affected when tachyzoites or host cells were only precoated with human Lf. Tanaka et al. [741] also demonstrated that bovine Lf inhibited in a concentration-dependent manner development of intracellular parasites. Supplement of apo-Lf and holo-Lf, but not transferrin, showed similar results. Mouse embryonal cells preincubated with Lf suppressed the intracellular growth of the parasite, while pretreatment of Lf to the macrophages did not show any inhibitory effects [741].
Finally, it must be noted that the trophozoites of T. gondii strain RH obtained from peritoneal exudate of infected mice treated with various concentrations of oolong tea and green tea showed that the lowest and effective concentration of tea to kill T. gondii completely was 0.5%, but the time necessary to kill the parasites was longer than a half hour [742].
Conclusion
In summary, physiologic swallowing of amniotic fluid containing maternal cells with T. gondii by the fetus may play an important role in early development of GT tract disturbances in patients with ASD or other GT diseases. The intestinal inflammation experimentally induced after the parasite infection shares a number of features in common with inflammatory bowel disease and necrotizing enterocolitis. Moreover, it seems that the morphometric abnormalities of the enteric nervous system found in animals caused by oral infection with T. gondii may markedly contribute for occurrence of the GT pathophysiology. NKT cells are critical for initiation of inflammatory bowel response against T. gondii, and transmission of the parasite from the infected dendritic cells to NKT cells probably plays an important role in this process because the enhanced density of dendritic T cells was demonstrated in the colon of autistic children with gastrointestinal disturbances. The presence of free tachyzoites and parasitophorus vacuoles inside the intestinal mast cells associated with marked morphological alterations in the cells may be responsible for the enteroglial-sustained gut inflammation and death of myenteric neurons in patients with IBD and several other GI tract abnormalities. It must be emphasized that significantly increased titers of anti-IgM and anti-IgG antibodies toward T. gondii have already been demonstrated in those individuals compared with controls. The changes in mast cells are similar to the recently proposed perpetuum mobilelike biomachinery causing persistent neuroinflammation in autistics, in which host-endoplasmic reticulum-parasitophorus vacuole interaction provides a route of entry for antigen presentation in T. gondii-infected dendritic cells. Mast cells secrete chemotactic factors able to recruite neutrophils, macrophages and lymphocytes when the parasite reaches the lamina propria, and finally reduce survival or cause death of myenteric neurons. The parasite may be also in charge for development of ileal and colonic pouches in patients with ulcerative colitis, Crohn’s disease, and in acute and chronic diverticular disease, because it causes post inflammatory damage to the enteric nervous system (ENS). This suggestion may be supported by the findings in laboratory animals that chronic oral/peritoneal inoculation with T. gondii genotypes I-III resulted in atrophy or hypoplasia of some segments of the GT and death/ hypertrophy of part of myenteric neurons. In addition, perinuclear antineutrophil cytoplasmic autoantibodies found consistently in the serum of children and adults with IBD may be associated with damage to myenteric neurons and marked changes in the cell body, nucleus and cytoplasm areas and nucleus/cell body area ratios depending on acute or chronic parasite infection. Virulence of T. gondii has been linked with strain-dependent distinct dendritic cell responses and reduced number of activated CD8+ T cells. Polymerase chain reaction analysis showed presence of the parasite only in the intestinal mucosa and submucosa, which suggested that the quantitative and morphometric alterations found in the myenteric neurons occurred as a result of profuse generation of NO and proinflammatory cytokines by the immune system during the infection. Thus, it seems that many congenital and acquired GI tract abnormalities and their final clinical presentation, including Hirschsprung’s disease, may be due to prenatal and/or postnatal damage of the ENS associated with peroral infection with T. gondii, its genotype, virulence/antigenicity, numbers of oocysts/ sporozoites/bradyzoites/tachyzoites, part of GI tract infected, and the host innate prenatal and postnatal immune state. On the other hand, it must be noted that lactoferrin (Lf) contained in the breast milk have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in the intestinal epithelial cells observed after oral T. gondii infection. Lf has the capacity to enter the nuclei of leukocytes and block the transcription factor NF-κB, which otherwise induces production of IL-1β, TNF-α, IL-6 and IL-8. Lf administered orally improved gastrointestinal morphology in growing calves because it enhanced size of Peyer’s patches in the ileum and decreased villous sizes in the jejunum. This effect of Lf and the hypertrophy of intestinal myenteric neurons demonstrated in rats inoculated with T. gondii may at least in part be responsible for development of LNH in some children with autism. In addition, Lf have immunomodulatory effects that were partly associated with LPS binding, and this may contribute for the delayed ASD diagnosis until approximately 1.5 to 2 years of age despite the evidence of prenatal morphological changes in the brain. Maturation of the innate immunity to T. gondii LPS antigens approximately at this age as compared with much earlier efficacious immune reaction to the parasite peptide antigens may strongly support this reasoning.
References
	White JF (2003) Intestinal pathophysiology in autism. Exp Biol Med (Maywood) 228: 639-649. Horvath K, Perman JA (2002) Autism and gastrointestinal symptoms. Curr Gastroenterol Rep 4: 251-258. Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, et al. (1998) Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet 351: 637-641. Wakefield AJ, Anthony A, Murch SH, Thomson M, Montgomery SM, et al. (2000) Enterocolitis in children with developmental disorders. Am J Gastroenterol 95: 2285-2295. D'Eufemia P, Celli M, Finocchiaro R, Pacifico L, Viozzi L, et al. (1996) Abnormal intestinal permeability in children with autism. Acta Paediatr 85: 1076-1079. Furlano RI, Anthony A, Day R, Brown A, McGarvey L, et al. (2001) Colonic CD8 and gamma delta T-cell infiltration with epithelial damage in children with autism. J Pediatr 138: 366-372. Torrente F, Ashwood P, Day R, Machado N, Furlano RI, et al. (2002) Small intestinal enteropathy with epithelial IgG and complement deposition in children with regressive autism. Mol Psychiatry 7: 375-382, 334. Horvath K, Papadimitriou JC, Rabsztyn A, Drachenberg C, Tildon JT (1999) Gastrointestinal abnormalities in children with autistic disorder. J Pediatr 135: 559-563. Ashwood P, Wakefield AJ (2006) Immune activation of peripheral blood and mucosal CD3+ lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms. J Neuroimmunol 173: 126-134. Gonzalez L, Lopez K, Navarro D, Negron L, Flores L, et al. (2006) Caracteristicas endoscopicas, histologicas e immnulogicas de la mucosa digestiva en ninos autistas con sintomas gastrointestinales. Archiv Venezol Peuricult Pediatr 69: 19-25. Gonzales L (2009) Gastrointestinal pathology in autism spectrum disorders: The Venezuelan experience. The Autism File Issue 32: 74-79. Buie T, Campbell DB, Fuchs GJ 3rd, Furuta GT, Levy J, et al. (2010) Evaluation, diagnosis, and treatment of gastrointestinal disorders in individuals with ASDs: a consensus report. Pediatrics 125 Suppl 1: S1-18. Mowat AM, Donachie AM, Parker LA, Robson NC, Beacock-Sharp H, et al. (2003) The role of dendritic cells in regulating mucosal immunity and tolerance. Novartis Found Symp 252: 291-302. de Theije CG, Wu J, da Silva SL, Kamphuis PJ, Garssen J, et al. (2011) Pathways underlying the gut-to-brain connection in autism spectrum disorders as future targets for disease management. Eur J Pharmacol 668 Suppl 1: S70-80. Goldszmid RS, Coppens I, Lev A, Caspar P, Mellman I, et al. (2009) Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii-infected dendritic cells. J Exp Med 206: 399-410. de Magistris L, Familiari V, Pascotto A, Sapone A, Frolli A, et al. (2010) Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. J Pediatr Gastroenterol Nutr 51: 418-424. Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B (2005) Dysregulated innate immune responses in young children with autism spectrum disorders: their relationship to gastrointestinal symptoms and dietary intervention. Neuropsychobiology 51: 77-85. McGinnis WR (2004) Oxidative stress in autism. Altern Ther Health Med 10: 22-36. Savidge TC, Newman P, Pothoulakis C, Ruhl A, Neunlist M, et al. (2007) Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione. Gastroenterology 132: 1344-1358. Von Boyen GBT, Schulte N, Pflüger C, Spaniol U, Hartmann C, et al. (2011) Distribution of enteric glia and GDNF during gut inflammation. Gastroenterology 11: 3. Chauhan A, Chauhan V (2006) Oxidative stress in autism. Pathophysiology 13: 171-181. Söğüt S, ZoroÄŸlu SS, Ozyurt H, Yilmaz HR, OzuÄŸurlu F, et al. (2003) Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism. Clin Chim Acta 331: 111-117. Salzman AL, Menconi MJ, Unno N, Ezzell RM, Casey DM, et al. (1995) Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers. Am J Physiol 268: G361-373. Unno N, Menconi MJ, Smith M, Fink MP (1995) Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial monolayers. Crit Care Med 23: 1170-1176. Wakefield AJ, Ashwood P, Limb K, Anthony A (2005) The significance of ileo-colonic lymphoid nodular hyperplasia in children with autistic spectrum disorder. Eur J Gastroenterol Hepatol 17: 827-836. Kokkonen J, Karttunen TJ (2002) Lymphonodular hyperplasia on the mucosa of the lower gastrointestinal tract in children: an indication of enhanced immune response? J Pediatr Gastroenterol Nutr 34: 42-46. Chauhan A, Mehta PD, Cohen LL, Barshatzky M, Brown WT, et al. (2006) Increased serum complement C3/C4 and alpha1-chymotrypsin levels in autism. In: International Meeting for Autism Research, Montreal, Canada. Chauhan A, Chauhan V, Cohen LL (2005) Increased serum complement C3 and C4 levels in autism: a correlation with severity. FEBS J 272: 492. Kushak RI, Lauwers GY, Winter HS, Buie TM (2011) Intestinal disaccharidase activity in patients with autism: effect of age, gender, and intestinal inflammation. Autism 15: 285-294. Yomogida S, Hua J, Sakamoto K, Nagaoka I (2008) Glucosamine suppresses interleukin-8 production and ICAM-1 expression by TNF-alpha-stimulated human colonic epithelial HT-29 cells. Int J Mol Med 22: 205-211. Wang L, Walia B, Evans J, Gewirtz AT, Merlin D, et al. (2003) IL-6 induces NF-kappa B activation in the intestinal epithelia. J Immunol 171: 3194-3201. Croonenberghs J, Bosmans E, Deboutte D, Kenis G, Maes M (2002) Activation of the inflammatory response system in autism. Neuropsychobiology 45: 1-6. Ashwood P, Van de Water J (2004) Is autism an autoimmune disease? Autoimmun Rev 3: 557-562. Singh VK (1996) Plasma increase of interleukin-12 and interferon-gamma. Pathological significance in autism. J Neuroimmunol 66: 143-145. Rossignol DA (2007) Hyperbaric oxygen therapy might improve certain pathophysiological findings in autism. Med Hypotheses 68: 1208-1227. Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA (2005) Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol 57: 67-81. Laurence JA, Fatemi SH (2005) Glial fibrillary acidic protein is elevated in superior frontal, parietal and cerebellar cortices of autistic subjects. Cerebellum 4: 206-210. Ahlsén G, Rosengren L, Belfrage M, Palm A, Haglid K, et al. (1993) Glial fibrillary acidic protein in the cerebrospinal fluid of children with autism and other neuropsychiatric disorders. Biol Psychiatry 33: 734-743. Ashwood P, Krakowiak P, Hertz-Picciotto I, Hansen R, Pessah I, et al. (2011) Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome. Brain Behav Immun 25: 40-45. Li X, Chauhan A, Sheikh AM, Patil S, Chauhan V, et al. (2009) Elevated immune response in the brain of autistic patients. J Neuroimmunol 207: 111-116. Jyonouchi H, Sun S, Le H (2001) Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression. J Neuroimmunol 120: 170-179. Ashwood P, Anthony A, Torrente F, Wakefield AJ (2004) Spontaneous mucosal lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms: mucosal immune activation and reduced counter regulatory interleukin-10. J Clin Immunol 24: 664-673. Okada K, Hashimoto K, Iwata Y, Nakamura K, Tsujii M, et al. (2007) Decreased serum levels of transforming growth factor-beta1 in patients with autism. Prog Neuropsychopharmacol Biol Psychiatry 31: 187-190. Molloy CA, Morrow AL, Meinzen-Derr J, Schleifer K, Dienger K, et al. (2006) Elevated cytokine levels in children with autism spectrum disorder. J Neuroimmunol 172: 198-205. Zoroglu SS, Yurekli M, Meram I, Sogut S, Tutkun H, et al. (2003) Pathophysiological role of nitric oxide and adrenomedullin in autism. Cell Biochem Funct 21: 55-60. Sweeten TL, Posey DJ, Shankar S, McDougle CJ (2004) High nitric oxide production in autistic disorder: a possible role for interferon-gamma. Biol Psychiatry 55: 434-437. Von Boyen GB, Steinkamp M, Reinshagen M, Schäfer KH, Adler G, et al. (2004) Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia. Gut 53: 222-228. Sweeten TL, Posey DJ, McDougle CJ (2003) High blood monocyte counts and neopterin levels in children with autistic disorder. Am J Psychiatry 160: 1691-1693. Fischer HG, Nitzgen B, Reichmann G, Hadding U (1997) Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells. Eur J Immunol 27: 1539-1548. Nagineni CN, Detrick B, Hooks JJ (2000) Toxoplasma gondii infection induces gene expression and secretion of interleukin 1 (IL-1), IL-6, granulocyte-macrophage colony-stimulating factor, and intercellular adhesion molecule 1 by human retinal pigment epithelial cells. Infect Immun 68: 407-410. Beaman MH, Hunter CA, Remington JS (1994) Enhancement of intracellular replication of Toxoplasma gondii by IL-6. Interactions with IFN-gamma and TNF-alpha. J Immunol 153: 4583-4587. Fang Y, Tan Y, Zhang Y, Li J (1999) [Effect of IL-6 on the multiplication of Toxoplasma gondii]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 17: 106-108. Malipiero U, Koedel U, Pfister HW, Levéen P, Bürki K, et al. (2006) TGFbeta receptor II gene deletion in leucocytes prevents cerebral vasculitis in bacterial meningitis. Brain 129: 2404-2415. Nagineni CN, Detrick B, Hooks JJ (2002) Transforming growth factor-beta expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis. Clin Exp Immunol 128: 372-378. Medical Reasearch Council (1999) Report of the strategy development group subgroup on research into inflammatory bowel disorders and autism. London: MRC. Quigley EM, Hurley D (2000) Autism and the gastrointestinal tract. Am J Gastroenterol 95: 2154-2156. Fernandez-Benares F, Salas A, Forne M, Esteve M, Espinos J, et al. (1999) Incidence of collagenous and lymphocytic colitis. A 5-year population-based study. Am J Gastroenterol 94: 418-423. McCashland TM, Donovan JP, Strobach RS, Linder J, Quigley EM (1992) Collagenous enterocolitis: a manifestation of gluten-sensitive enteropathy. J Clin Gastroenterol 15: 45-51. Goyal RK, Hirano I (1996) The enteric nervous system. N Engl J Med 334: 1106-1115. Cabarrocas J, Savidge TC, Liblau RS (2003) Role of enteric glial cells in inflammatory bowel disease. Glia 41: 81-93. Rühl A (2006) Glial regulation of neuronal plasticity in the gut: implications for clinicians. Gut 55: 600-602. Taylor CT, Keely SJ (2007) The autonomic nervous system and inflammatory bowel disease. Auton Neurosci 133: 104-114. Wedel T, Roblick U, Gleiss J, Schiedeck T, Bruch HP, et al. (1999) Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus. Ann Anat 181: 327-337. Schemann M, Neunlist M (2004) The human enteric nervous system. Neurogastroenterol Motil 1: 55-59. Hoff S, Zeller F, von Weyhern CW, Wegner M, Schemann M, et al. (2008) Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti-Sox8/9/10 antibody. J Comp Neurol 509: 356-371. Cirillo C, Sarnelli G, Esposito G, Turco F, Steardo L, et al. (2011) S100B protein in the gut: the evidence for enteroglial-sustained intestinal inflammation. World J Gastroenterol 17: 1261-1266. Driessen A, Creemers J, Geboes K (1995) Anti-Leu-19 is a marker for nervous tissue in the mucosa of the human rectum. Acta Anat (Basel) 153: 127-134. Gershon MD (2005) Nerves, reflexes, and the enteric nervous system: pathogenesis of the irritable bowel syndrome. J Clin Gastroenterol 39: S184-193. Jessen KR (2004) Glial cells. Int J Biochem Cell Biol 36: 1861-1867. Komuro T, BaÅ‚uk P, Burnstock G (1982) An ultrastructural study of neurons and non-neuronal cells in the myenteric plexus of the rabbit colon. Neuroscience 7: 1797-1806. Furness JB (2000) Types of neurons in the enteric nervous system. J Auton Nerv Syst 81: 87-96. Gabella G (1972) Fine structure of the myenteric plexus in the guinea-pig ileum. J Anat 111: 69-97. Gabella G (1982) On the ultrastructure of the enteric nerve ganglia. Scand J Gastroenterol Suppl 71: 15-25. Gabella G (1994) Structure of muscles and nerves in the gastrointestinal tract. In: Johnson LR, Alpers DH, Jacobson ED, Walsh JH (eds), Physiology of the Gastrointestinal Tract (3rdedn), Raven Press, New York 751-793. Furness JB, Clerc N, Vogalis F, Stebbing MJ (2003) The enteric nervous system and its extrinsic connections. Yamada T, Alpers DH (eds). Textbook of Gastroenterology, Lippincott Williams, Philadelphia 12-34. Brookes SJ (2001) Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec 262: 58-70. Gershon MD, Kirchgessner AL, Wade PR (1994) Functional anatomy of the enteric nervous system. Johnson LR, Alpers DH, Jacobson ED, Walsh JH (eds), Physiology of the Gastrointestinal Tract (3rdedn), New York , Raven Press 381-422. Sharkey KA, Lomax AE (2002) Structure and function of the enteric nervous system: neurological disease and its consequences for neuromuscular function in the gastrointestinal tract. Brown WF, Bolton CF, Amimoff MJ (eds), Clinical Neurophysiology and Neuromuscular Diseases, WB Saunders, Philadelphia 527-555. Lomax AE, Fernández E, Sharkey KA (2005) Plasticity of the enteric nervous system during intestinal inflammation. Neurogastroenterol Motil 17: 4-15. Gabella G, Trigg P (1984) Size of neurons and glial cells in the enteric ganglia of mice, guinea-pigs, rabbits and sheep. J Neurocytol 13: 49-71. Hanani M, Zamir O, Baluk P (1989) Glial cells in the guinea pig myenteric plexus are dye coupled. Brain Res 497: 245-249. Nagahama M, Semba R, Tsuzuki M, Aoki E (2001) L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum. Biol Signals Recept 10: 336-340. Fletcher EL, Clark MJ, Furness JB (2002) Neuronal and glial localization of GABA transporter immunoreactivity in the myenteric plexus. Cell Tissue Res 308: 339-346. Hounnou G, Destrieux C, Desmé J, Bertrand P, Velut S (2002) Anatomical study of the length of the human intestine. Surg Radiol Anat 24: 290-294. Schäfer KH, Hänsgen A, Mestres P (1999) Morphological changes of the myenteric plexus during early postnatal development of the rat. Anat Rec 256: 20-28. Marese AC, de Freitas P, Natali MR (2007) Alterations of the number and the profile of myenteric neurons of Wistar rats promoted by age. Auton Neurosci 137: 10-18. Gabella G (1971) Neuron size and number in the myenteric plexus of the newborn and adult rat. J Anat 109: 81-95. Phillips RJ, Powley TL (2001) As the gut ages: timetables for aging of innervation vary by organ in the Fischer 344 rat. J Comp Neurol 434: 358-377. Meciano Filho J, Carvalho VC, de Souza RR (1995) Nerve cell loss in the myenteric plexus of the human esophagus in relation to age: a preliminary investigation. Gerontology 41: 18-21. Gomes OA, de Souza RR, Liberti EA (1997) A preliminary investigation of the effects of aging on the nerve cell number in the myenteric ganglia of the human colon. Gerontology 43: 210-217. Wade PR (2002) Aging and neural control of the GI tract. I. Age-related changes in the enteric nervous system. Am J Physiol Gastrointest Liver Physiol 283: G489-495. Thrasivoulou C, Ridha H, Soubeyre V, Hoyle CHV, Saffrey MJ, et al. (2000) Free radical buffering in rat enteric neurons: effects of age and neurotrophic factors. Eur J Neurosci 12: 33. Johnson RJ, Schemann M, Santer RM, Cowen T (1998) The effects of age on the overall population and on sub-populations of myenteric neurons in the rat small intestine. J Anat 192 : 479-488. Phillips RJ, Kieffer EJ, Powley TL (2003) Aging of the myenteric plexus: neuronal loss is specific to cholinergic neurons. Auton Neurosci 106: 69-83. Wade PR, Cowen T (2004) Neurodegeneration: a key factor in the ageing gut. Neurogastroenterol Motil 16 Suppl 1: 19-23. Newgreen D, Young HM (2002) Enteric nervous system: Development and developmental disturbances–part 2. Pediatr Dev Pathol 5: 329-349. Gershon MD (2000) Functional anatomy of the enteric nervous system: A developmental perspective relevant to the pathogenesis of Hirschsprung’s disease. Holschneider AM, Puri P (eds) Hirschprung’s Disease and Allied Disorders, Harwood Academic Publishers, Amsterdam, The Nederlands, 19-58. Costa M, Brookes SJ, Hennig GW (2000) Anatomy and physiology of the enteric nervous system. Gut 47 Suppl 4: iv15-19. Van Laere K, Vonck K, Boon P, Brans B, Vandekerckhove T, et al. (2000) Vagus nerve stimulation in refractory epilepsy: SPECT activation study. J Nucl Med 41: 1145-1154. Binnie CD (2000) Vagus nerve stimulation for epilepsy: a review. Seizure 9: 161-169. Rush AJ, George MS, Sackeim HA, Marangell LB, Husain MM, et al. (2000) Vagus nerve stimulation (VNS) for treatment-resistant depressions: A multicenter study. Biol Psychiatry 47: 276-286. Rosenbaum JF, Heninger G (2000) Vagus nerve stimulation for treatment-resistant depression. Biol Psychiatry 47: 273-275. Clark KB, Naritoku DK, Smith DC, Browning RA, Jensen RA (1999) Enhanced recognition memory following vagus nerve stimulation in human subjects. Nat Neurosci 2: 94-98. Gershon MD, Ratcliffe EM (2004) Developmental biology of the enteric nervous system: pathogenesis of Hirschsprung's disease and other congenital dysmotilities. Semin Pediatr Surg 13: 224-235. Schreiner M, Liesenfeld O (2009) Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature. Mem Inst Oswaldo Cruz 104: 221-233. Rattan J, Hallak A, Shvartzman H, Felner S, Gilat T (1986) Sabin-Feldman dye test in ulcerative colitis and Crohn's disease. Dis Colon Rectum 29: 402-404. Bush TG, Savidge TC, Freeman TC, Cox HJ, Campbell EA, et al. (1998) Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 93: 189-201. Cornet A, Savidge TC, Cabarrocas J, Deng WL, Colombel JF, et al. (2001) Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease? Proc Natl Acad Sci U S A 98: 13306-13311. Edelson MB, Bagwell CE, Rozycki HJ (1999) Circulating pro- and counterinflammatory cytokine levels and severity in necrotizing enterocolitis. Pediatrics 103: 766-771. Harris MC, D'Angio CT, Gallagher PR, Kaufman D, Evans J, et al. (2005) Cytokine elaboration in critically ill infants with bacterial sepsis, necrotizing entercolitis, or sepsis syndrome: correlation with clinical parameters of inflammation and mortality. J Pediatr 147: 462-468. Caplan MS, Simon D, Jilling T (2005) The role of PAF, TLR, and the inflammatory response in neonatal necrotizing enterocolitis. Semin Pediatr Surg 14: 145-151. Upperman JS, Potoka D, Grishin A, Hackam D, Zamora R, et al. (2005) Mechanisms of nitric oxide-mediated intestinal barrier failure in necrotizing enterocolitis. Semin Pediatr Surg 14: 159-166. Hsueh W, Caplan MS, Qu XW, Tan XD, De Plaen IG, et al. (2003) Neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts. Pediatr Dev Pathol 6: 6-23. Claud EC, Walker WA (2001) Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis. FASEB J 15: 1398-1403. Lin PW, Nasr TR, Stoll BJ (2008) Necrotizing enterocolitis: recent scientific advances in pathophysiology and prevention. Semin Perinatol 32: 70-82. Claud EC, Savidge T, Walker WA (2003) Modulation of human intestinal epithelial cell IL-8 secretion by human milk factors. Pediatr Res 53: 419-425. Rühl A, Nasser Y, Sharkey KA (2004) Enteric glia. Neurogastroenterol Motil 16: 44-49. Mawe GM, Collins SM, Shea-Donohue T (2004) Changes in enteric neural circuitry and smooth muscle in the inflamed and infected gut. Neurogastroenterol Motil 16 Suppl 1: 133-136. Der T, Bercik P, Donnelly G, Jackson T, Berezin I, et al. (2000) Interstitial cells of cajal and inflammation-induced motor dysfunction in the mouse small intestine. Gastroenterology 119: 1590-1599. Linden DR, Chen JX, Gershon MD, Sharkey KA, Mawe GM (2003) Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol 285: G207-216. Lu G, Qian X, Berezin I, Telford GL, Huizinga JD, et al. (1997) Inflammation modulates in vitro colonic myoelectric and contractile activity and interstitial cells of Cajal. Am J Physiol 273: G1233-1245. Brown DR, Timmermans JP (2004) Lessons from the porcine enteric nervous system. Neurogastroenterol Motil 16 Suppl 1: 50-54. Furness JB, Costa M (1987) The Enteric Nervous System, Churchill Livingstone, Edinburgh. Rühl A, Franzke S, Stremmel W (2001) IL-1beta and IL-10 have dual effects on enteric glial cell proliferation. Neurogastroenterol Motil 13: 89-94. Rühl A, Franzke S, Collins SM, Stremmel W (2001) Interleukin-6 expression and regulation in rat enteric glial cells. Am J Physiol Gastrointest Liver Physiol 280: G1163-1171. Murakami M, Ohta T, Ito S (2009) Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells. J Neurosci Res 87: 2095-2104. Cirillo C, Sarnelli G, Mango A, Esposito I, Cuomo R (2009) 30 Effect of pro-inflammatory stimuli on cellular activation and nitric oxide production in primary cultures of human enteric glia. Gastroenterology 136: A4. Pardo CA, Vargas DL, Zimmerman AW (2005) Immunity, neuroglia and neuroinflammation in autism. Int Rev Psychiatry 17: 485-495. Matowicka-Karna J, Dymicka-Piekarska V, Kemona H (2009) Does Toxoplasma gondii infection affect the levels of IgE and cytokines (IL-5, IL-6, IL-10, IL-12, and TNF-alpha)? Clin Dev Immunol 2009: 374696. Sharkey KA, Parr EJ (1996) The enteric nervous system in intestinal inflammation. Canadian Journal of Gastroenterology 10: 335-341. Törnblom H, Lindberg G, Nyberg B, Veress B (2002) Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome. Gastroenterology 123: 1972-1979. De Giorgio R, Barbara G, Stanghellini V, De Ponti F, Salvioli B, et al. (2002) Clinical and morphofunctional features of idiopathic myenteric ganglionitis underlying severe intestinal motor dysfunction: a study of three cases. Am J Gastroenterol 97: 2454-2459. Veress B, Nyberg B, Törnblom H, Lindberg G (2009) Intestinal lymphocytic epithelioganglionitis: a unique combination of inflammation in bowel dysmotility: a histopathological and immunohistochemical analysis of 28 cases. Histopathology 54: 539-549. Geboes K, Collins S (1998) Structural abnormalities of the nervous system in Crohn's disease and ulcerative colitis. Neurogastroenterol Motil 10: 189-202. Villanacci V, Bassotti G, Nascimbeni R, Antonelli E, Cadei M, et al. (2008) Enteric nervous system abnormalities in inflammatory bowel diseases. Neurogastroenterol Motil 20: 1009-1016. Ohlsson B, Veress B, Lindgren S, Sundkvist G (2007) Enteric ganglioneuritis and abnormal interstitial cells of Cajal: features of inflammatory bowel disease. Inflamm Bowel Dis 13: 721-726. Wang XY, Zarate N, Soderholm JD, Bourgeois JM, Liu LW, et al. (2007) Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease. Neurogastroenterol Motil 19: 349-364. Rumessen JJ (1996) Ultrastructure of interstitial cells of Cajal at the colonic submuscular border in patients with ulcerative colitis. Gastroenterology 111: 1447-1455. Crohn BB, Oppenheimer GD (1932) Regional ileitis: a pathologic and clinical entity. JAMA 99: 1323-1329. HAFERKAMP O (1959) [On productive inflammation in enteritis regionalis (Crohn)]. Beitr Pathol Anat 121: 27-38. ANTONIUS JI, GUMP FE, LATTES R, LEPORE M (1960) A study of certain microscopic features in regional enteritis, and their possible prognostic significance. Gastroenterology 38: 889-905. Mottet NK (1971) Histopathologic spectrum of regional enteritis and ulcerative colitis. Major Probl Pathol 2: 1-249. Strobach RS, Ross AH, Markin RS, Zetterman RK, Linder J (1990) Neural patterns in inflammatory bowel disease: an immunohistochemical survey. Mod Pathol 3: 488-493. Geboes K, Rutgeerts P, Penninckx F, Desmet V, Vantrappen G (1989) The destruction of the autonomic nervous system in Crohn’s disease is due to immunologic effector cells. Gastroenterology 96: A168. Cook MG, Dixon MF (1973) An analysis of the reliability of detection and diagnostic value of various pathological features in Crohn's disease and ulcerative colitis. Gut 14: 255-262. Riemann JF, Schmidt H (1982) Ultrastructural changes in the gut autonomic nervous system following laxative abuse and in other conditions. Scand J Gastroenterol Suppl 71: 111-124. Geboes K (1993) Immunopathological studies of the small intestinal intramural nervous system and of intramural vessels in Crohn's disease. Verh K Acad Geneeskd Belg 55: 267-301. Nadorra R, Landing BH, Wells TR (1986) Intestinal plexuses in Crohn's disease and ulcerative colitis in children: pathologic and microdissection studies. Pediatr Pathol 6: 267-287. Dvorak AM, Osage JE, Monahan RA, Dickersin GR (1980) Crohn’s disease: transmission electron microscopic studies. III. Target tissues proliferation of and injury to smooth muscle and the autonomic nervous system. Human Pathol 11: 620-634. Steinhoff MM, Kodner IJ, DeSchryver-Kecskemeti K (1988) Axonal degeneration/necrosis: a possible ultrastructural marker for Crohn's disease. Mod Pathol 1: 182-187. Brewer DB, Thompson H, Haynes IG, Alexander-Williams J (1990) Axonal damage in Crohn's disease is frequent, but non-specific. J Pathol 161: 301-311. Dvorak AM (1988) Ultrastructural pathology of Crohn’s disease. In: Goebell H, Peskar BM, Malchow H (eds), Inflammatory Bowel Diseases, MTP Press Ltd, Lancaster, Boston 3-42. OKAMOTO E, KAKUTANI T, IWASAKI T, NAMBA M, UEDA T (1964) MORPHOLOGICAL STUDIES ON THE MYENTERIC PLEXUS OF THE COLON IN CHRONIC ULCERATIVE COLITIS. A PRELIMINARY REPORT. Med J Osaka Univ 15: 85-106. van der Zypen E (1965) [Light and electron microscopic findings on the autonomic nervous system of the colon in human ulcerative colitis]. Dtsch Z Nervenheilkd 187: 787-836. Siemers PT, Dobbins WO 3rd (1974) The Meissner plexus in Crohn's disease of the colon. Surg Gynecol Obstet 138: 39-42. Van Patter WN, Bargen JA, Dockerty MC, Feldmann WH, Mayo CW, et al. (1954) Regional enteritis. Gastroenterology 26: 347-351. DAVIS DR, DOCKERTY MB, MAYO CW (1955) The myenteric plexus in regional enteritis: a study of the number of ganglion cells in the ileum in 24 cases. Surg Gynecol Obstet 101: 208-216. Bishop AE, Polak JM, Bryant MG, Bloom SR, Hamilton S (1980) Abnormalities of vasoactive intestinal polypeptide-containing nerves in Crohn's disease. Gastroenterology 79: 853-860. Belai A, Boulos PB, Robson T, Burnstock G (1997) Neurochemical coding in the small intestine of patients with Crohn's disease. Gut 40: 767-774. Oehmichen M, Reifferscheid P (1977) Intramural ganglion cell degeneration in inflammatory bowel disease. Digestion 15: 482-496. O'Morain C, Bishop AE, McGregor GP, Levi AJ, Bloom SR, et al. (1984) Vasoactive intestinal peptide concentrations and immunocytochemical studies in rectal biopsies from patients with inflammatory bowel disease. Gut 25: 57-61. Sjölund K, Schaffalitzky OB, Muckadell DE, Fahrenkrug J, Håkanson R, et al. (1983) Peptide-containing nerve fibres in the gut wall in Crohn's disease. Gut 24: 724-733. Kubota Y, Petras RE, Ottaway CA, Tubbs RR, Farmer RG, et al. (1992) Colonic vasoactive intestinal peptide nerves in inflammatory bowel disease. Gastroenterology 102: 1242-1251. Kubota Y, Petras RE, Tubbs RR, Farmer RG, Fiocchi C, et al. (1991) Loss of vasoactive intestinal peptide (VIP) immunoreactive nerve in fibers in the colon of IBD patients. Tsuchiya M, Nagura H, Hibi T, Moro I (eds), Frontiers of Mucosal Immunology, Excerpta Medica, Amsterdam 843-846. Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, et al. (1992) Major histocompatibility class II expression on the small intestinal nervous system in Crohn's disease. Gastroenterology 103: 439-447. Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, et al. (1992) Major histocompatibility class II expression on the small intestinal nervous system in Crohn's disease. Gastroenterology 103: 439-447. Koretz K, Momburg F, Otto HF, Möller P (1987) Sequential induction of MHC antigens on autochthonous cells of ileum affected by Crohn's disease. Am J Pathol 129: 493-502. Von Herbay A, Castrucci M, Otten U, Otto HF (1993) Coexpression of nerve growth factor receptor and cytokine receptor CD27 in enteric neurons and immune cells in ulcerative colitis and Crohn’s disease. Gastroenterology 104: A797. Vasina V, Barbara G, Talamonti L, Stanghellini V, Corinaldesi R, et al. (2006) Enteric neuroplasticity evoked by inflammation. Auton Neurosci 126-127: 264-72. Neunlist M, Aubert P, Toquet C, Oreshkova T, Barouk J, et al. (2003) Changes in chemical coding of myenteric neurones in ulcerative colitis. Gut 52: 84-90. Schneider J, Jehle EC, Starlinger MJ, Neunlist M, Michel K, et al. (2001) Neurotransmitter coding of enteric neurones in the submucous plexus is changed in non-inflamed rectum of patients with Crohn's disease. Neurogastroenterol Motil 13: 255-264. Ekblad E, Sjuve R, Arner A, Sundler F (1998) Enteric neuronal plasticity and a reduced number of interstitial cells of Cajal in hypertrophic rat ileum. Gut 42: 836-844. Ekblad E, Bauer AJ (2004) Role of vasoactive intestinal peptide and inflammatory mediators in enteric neuronal plasticity. Neurogastroenterol Motil 16 Suppl 1: 123-128. Roberts PJ, Morgan K, Miller R, Hunter JO, Middleton SJ (2001) Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease. Gut 48: 468-472. Renzi D, Pellegrini B, Tonelli F, Surrenti C, Calabrò A (2000) Substance P (neurokinin-1) and neurokinin A (neurokinin-2) receptor gene and protein expression in the healthy and inflamed human intestine. Am J Pathol 157: 1511-1522. Arnold SJ, Facer P, Yiangou Y, Chen MX, Plumpton C, et al. (2003) Decreased potassium channel IK1 and its regulator neurotrophin-3 (NT-3) in inflamed human bowel. Neuroreport 14: 191-195. Yiangou Y, Facer P, Smith JA, Sangameswaran L, Eglen R, et al. (2001) Increased acid-sensing ion channel ASIC-3 in inflamed human intestine. Eur J Gastroenterol Hepatol 13: 891-896. Yiangou Y, Facer P, Baecker PA, Ford AP, Knowles CH, et al. (2001) ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease. Neurogastroenterol Motil 13: 365-369. Geller SA, Cohen A (1983) Arterial inflammatory-cell infiltration in Crohn's disease. Arch Pathol Lab Med 107: 473-475. Wakefield AJ, Sankey EA, Dhillon AP, Sawyerr AM, More L, et al. (1991) Granulomatous vasculitis in Crohn's disease. Gastroenterology 100: 1279-1287. STORSTEEN KA, KERNOHAN JW, BARGEN JA (1953) The myenteric plexus in chronic ulcerative colitis. Surg Gynecol Obstet 97: 335-343. Dvorak AM, Osage JE, Monahan RA, Dickersin GR (1980) Crohn’s disease: transmission electron microscopic studies. Human Pathol 11: 620-634. Bradley JS Jr, Parr EJ, Sharkey KA (1997) Effects of inflammation on cell proliferation in the myenteric plexus of the guinea-pig ileum. Cell Tissue Res 289: 455-461. Szabó V, Fehér E (1991) Ultrastructural changes in the nerve elements in Crohn's disease. Acta Chir Hung 32: 25-32. Rühl A (2005) Glial cells in the gut. Neurogastroenterol Motil 17: 777-790. Bassotti G, Villanacci V, Fisogni S, Rossi E, Baronio P, et al. (2007) Enteric glial cells and their role in gastrointestinal motor abnormalities: introducing the neuro-gliopathies. World J Gastroenterol 13: 4035-4041. Bassotti G, Villanacci V, Antonelli E, Morelli A, Salerni B (2007) Enteric glial cells: new players in gastrointestinal motility? Lab Invest 87: 628-632. Hirata I, Berrebi G, Austin LL, Keren DF, Dobbins WO 3rd (1986) Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease. Dig Dis Sci 31: 593-603. Chadwick VS, Chen W, Shu D, Paulus B, Bethwaite P, et al. (2002) Activation of the mucosal immune system in irritable bowel syndrome. Gastroenterology 122: 1778-1783. Ogra PL (2010) Ageing and its possible impact on mucosal immune responses. Ageing Res Rev 9: 101-106. Wilson CB, Kollmann TR (2008) Induction of antigen-specific immunity in human neonates and infants. Nestle Nutr Workshop Ser Pediatr Program 61: 183-195. MacDonald TT, Spencer J (1990) Ontogeny of the mucosal immune response. Springer Semin Immunopathol 12: 129-137. MacDonald TT, Spencer J (1994) Ontogeny of the gut-associated lymphoid system in man. Acta Paediatr Suppl 83: 3-5. Cornes JS (1965) Number, size, and distribution of Peyer’s patches in the human small intestine. Part I. The development of Peyer’s patches. Gut 6: 225-229. Jónsdóttir I (2007) Maturation of mucosal immune responses and influence of maternal antibodies. J Comp Pathol 137 Suppl 1: S20-26. Wuorimaa T, Käyhty H (2002) Current state of pneumococcal vaccines. Scand J Immunol 56: 111-129. Kollmann TR, Crabtree J, Rein-Weston A, Blimkie D, Thommai F, et al. (2009) Neonatal innate TLR-mediated responses are distinct from those of adults. J Immunol 183: 7150-7160. Corbett NP. Blimkie D, Ho K, Cai B, Sutherland DP, et al. (2010) Ontogeny of toll-like receptor mediated cytokine responses of human blood mononuclear cells. PLoS One 5: e15041. Minns LA, Menard LC, Foureau DM, Darche S, Ronet C, et al. (2006) TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii. J Immunol 176: 7589-7597. Dimier-Poisson I, Aline F, Mévélec MN, Beauvillain C, Buzoni-Gatel D, et al. (2003) Protective mucosal Th2 immune response against Toxoplasma gondii by murine mesenteric lymph node dendritic cells. Infect Immun 71: 5254-5265. Furuta T, Kikuchi T, Akira S, Watanabe N, Yoshikawa Y (2006) Roles of the small intestine for induction of toll-like receptor 4-mediated innate resistance in naturally acquired murine toxoplasmosis. Int Immunol 18: 1655-1662. Buzoni-Gatel D, Werts C (2006) Toxoplasma gondii and subversion of the immune system. Trends Parasitol 22: 448-452. Lüder CG, Lang T, Beuerle B, Gross U (1998) Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii. Clin Exp Immunol 112: 308-316. Silva LS, Sartori AL, Zaniolo LM, da Silva AV, Sant'Ana Dde M, et al. (2011) Toxoplasma gondii: myenteric neurons of intraperitoneally inoculated rats show quantitative and morphometric alterations. Exp Parasitol 129: 5-10. Sugauara EY, Sant'Ana Dde M, Almeida EC, Reis AB, Silva AV, et al. (2008) Alterations of the myenteric plexus of the ileum and the descending colon caused by Toxoplasma gondii (genotype III). Arq Neuropsiquiatr 66: 516-523. da Silva Pde C, Shiraishi CS, Silva AV, Gonçalves GF, Sant'Ana Dde M, et al. (2010) Toxoplasma gondii: a morphometric analysis of the wall and epithelial cells of pigs intestine. Exp Parasitol 125: 380-383. Odorizzi L, Moreira NM, Gonçalves GF, da Silva AV, Sant'ana Dde M, et al. (2010) Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis. Auton Neurosci 155: 68-72. Pereira LS, da Silva AV, Araujo EJA, Sant’Ana DMG (2010) Hypertrophy of NADPH-diaphorase positive myenteric neurons in rat jejunum after acute infection caused by Toxoplasma gondii. J Venom Anim Toxins Tropical Dis 16: 298-310. Alves MS, da Silva AV, Bianchi LRO, Araujo EJA, Sant’Ana DMG (2011) Toxoplasma gondii induces death of gastric myenteric neurons in rats. Int J Morphol 29: 293-298. Prandota J. Increased generation of antibodies and autoantibodies directed against brain proteins in patients with autism and their families may be caused by T. gondii infection. Maternal and fetal miocrochimerisms probably play an important role in these processes acting as a “Trojan horse” in dissemination of the parasite. Gemma C (eds) Neuroinflammation: Pathogenesis, Mechanisms and Management, Nova Publishers, New York 2012. Sugauara EYY, Sant’Ana DMG, da Silva AV, de Souza EA, de Almeida Araujo EJ (2009) Hypertrophy of the neurons in the ileum of rats infected with cysts of Toxoplasma gondii (genotype II). Acta Scient Biol Sci (UEM). Soares J, Moreira NM, da Silva AV, Sant'Ana Dde M, Araújo EJ (2009) [Chronic infection due to Toxoplasma gondii inducing neuron hypertrophy of the myenteric plexus of Rattus norvegicus descending colon]. Rev Bras Parasitol Vet 18: 57-60. Berenreiterová M, Flegr J, KubÄ›na AA, NÄ›mec P (2011) The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis. PLoS One 6: e28925. Seva AP, da Silva RC, da Silva AV, De Castro APB, Menozzi BD, et al. (2006) Avaliacao da virulencia de cepas de Toxoplasma gondii, em camundongos, isolados de caes com sinais neurologicos, em Botucatu, SP. Vet Zootec 13: 33-43. Howe DK, Sibley LD (1995) Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis 172: 1561-1566. Grigg ME, Ganatra J, Boothroyd JC, Margolis TP (2001) Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J Infect Dis 184: 633-639. Carme B, Demar M, Ajzenberg D, Dardé ML (2009) Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana. Emerg Infect Dis 15: 656-658. Dubey JP (1996) Strategies to reduce transmission of Toxoplasma gondii to animals and humans. Vet Parasitol 64: 65-70. Freyre A, Correa O, Falcon J, Mendez J, Gonzalez M, et al. (2001) Some factors influencing transmission of Toxoplasma in pregnant rats fed cysts. Parasitol Res 87: 941-944. Dubey JP, Navarro IT, Graham DH, Dahl E, Freire RL, et al. (2003) Characterization of Toxoplasma gondii isolates from free range chickens from Parana, Brazil. Vet Parasitol 117: 229-234. Jacobs L, Remington JS, Melton ML (1960) A survey of meat samples from swine, cattle, and sheep for the presence of encysted Toxoplasma. J Parasitol 46: 23-28. Tait ED, Jordan KA, Dupont CD, Harris TH, Gregg B, et al. (2010) Virulence of Toxoplasma gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells. J Immunol 185: 1502-1512. Gregg B, Dzierszinski F, Tait E, Jordan KA, Hunter CA, et al. (2011) Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii. PLoS One 6: e22936. Barragan A, Sibley LD (2003) Migration of Toxoplasma gondii across biological barriers. Trends Microbiol 11: 426-430. Kim L, Butcher BA, Lee CW, Uematsu S, Akira S, et al. (2006) Toxoplasma gondii genotype determines MyD88-dependent signaling in infected macrophages. J Immunol 177: 2584-2591. Robben PM, Mordue DG, Truscott SM, Takeda K, Akira S, et al. (2004) Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype. J Immunol 172: 3686-3694. Benson A, Pifer R, Behrendt CL, Hooper LV, Yarovinsky F (2009) Gut commensal bacteria direct a protective immune response against Toxoplasma gondii. Cell Host Microbe 6: 187-196. Torres JRP (2008) Perda e plasticidade neuronal mienterica no jejuno de ratos infectados por taquizoitos de Toxoplasma gondii. 40 f. Dissertacao (Mestrado em Ciencia Animal), Diretoria Executiva de Gestao de Pesquisa e da Pos-Graduacao, Universidade Paranaense – UNIPAR. Bonapaz RS, Hermes-Uliana C, Santos FN, da Silva AV, Araujo EJA, et al. (2010) Effects of infection with Toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus). Pesq Vet Bras 30: 787-792. Weiss LM, Kim K, Templeton TJ, Chaudhary K, Fox BA, et al. (2007) Toxoplasma gondii. The Model Apicomplexan. Perspectives and Methods. Weiss LM, Kim K (eds) Academic Press, Elsevier, London, Burlington, San Diego, Rio de Janeiro, 341-366. Arantes RM, Marche HH, Bahia MT, Cunha FQ, Rossi MA, et al. (2004) Interferon-gamma-induced nitric oxide causes intrinsic intestinal denervation in Trypanosoma cruzi-infected mice. Am J Pathol 164: 1361-1368. Miranda Neto MH, Molinari SL, Natali MR, Sant'Ana DM (2001) Regional differences in the number and type of myenteric neurons of the ileum of rats: a comparison of techniques of the neuronal evidentiation. Arq Neuropsiquiatr 59: 54-59. Chardès T, Velge-Roussel F, Mevelec P, Mevelec MN, Buzoni-Gatel D, et al. (1993) Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice. Immunology 78: 421-429. Akiho H, Ihara E, Motomura Y, Nakamura K (2011) Cytokine-induced alterations of gastrointestinal motility in gastrointestinal disorders. World J Gastrointest Pathophysiol 2: 72-81. Kasper LH, Buzoni-Gatel D (2001) Ups and downs of mucosal cellular immunity against protozoan parasites. Infect Immun 69: 1-8. Speer CA, Dubey JP (1998) Ultrastructure of early stages of infections in mice fed Toxoplasma gondii oocysts. Parasitology 116 : 35-42. Skorich DN, Chiappino ML, Nichols BA (1988) Invasion of the guinea pig conjunctiva by Toxoplasma gondii. Invest Ophthalmol Vis Sci 29: 1871-1880. Alpert L, Miller M, Alpert E, Satin R, Lamoureux E, et al. (1996) Gastric toxoplasmosis in acquired immunodeficiency syndrome: antemortem diagnosis with histopathologic characterization. Gastroenterology 110: 258-264. Kofman E, Khorsandi A, Sarlin J, Adhami K (1996) Gastric toxoplasmosis: case report and review of the literature. Am J Gastroenterol 91: 2436-2438. Smart PE, Weinfeld A, Thompson NE, Defortuna SM (1990) Toxoplasmosis of the stomach: a cause of antral narrowing. Radiology 174: 369-370. Montoya JG, Remington JS (2000) Toxoplasma gondii. Mandell GL, Bennett JE, Dolin R (eds) Principles and Practice of Infectious Diseases (5thed) Churchill Livingstone, Philadelphia 2858-2888. Ganji M, Tan A, Maitar MI, Weldon-Linne CM, Weisenberg E, et al. (2003) Gastric toxoplasmosis in a patient with acquired immunodeficiency syndrome. A case report and review of the literature. Arch Pathol Lab Med 127: 732-734. Merzianu M, Gorelick SM, Paje V, Kotler DP, Sian C (2005) Gastric toxoplasmosis as the presentation of acquired immunodeficiency syndrome. Arch Pathol Lab Med 129: e87-90. Pauwels A, Meyohas MC, Eliaszewicz M, Legendre C, Mougeot G, et al. (1992) Toxoplasma colitis in the acquired immunodeficiency syndrome. Am J Gastroenterol 87: 518-519. Yang M, Perez E (1995) Disseminated toxoplasmosis as a cause of diarrhea. South Med J 88: 860-861. Lowe D, Hessler R, Lee J, Schade R, Chaudhary A (2006) Toxoplasma colitis in a patient with acquired immune deficiency syndrome. Gastrointest Endosc 63: 341-342. Frenkel JK (1997) Toxoplasmosis. Connor DN, Chandler FW, Manz HJ, Schwartz DA, Lack EE (eds), Pathology of Infectious Diseases, Appleton & Lange, Stamford, 1261-1278. Canfield PJ, Hartley WJ, Dubey JP (1990) Lesions of toxoplasmosis in Australian marsupials. J Comp Pathol 103: 159-167. Cunningham AA, Buxton D, Thomson KM (1992) An epidemic of toxoplasmosis in a captive colony of squirrel monkeys (Saimiri sciureus). J Comp Pathol 107: 207-219. Hartley WJ, Dubey JP, Spielman DS (1990) Fatal toxoplasmosis in koalas (Phascolarctos cinereus). J Parasitol 76: 271-272. McConnell JF, Sparkes AH, Blunden AS, Neath PJ, Sansom J (2007) Eosinophilic fibrosing gastritis and toxoplasmosis in a cat. J Feline Med Surg 9: 82-88. Florêncio FR, Albuquerque Filho FB, Moraes MA (1992) [Toxoplasma gondii in the gastric mucosa as the first finding in an AIDS patient]. Rev Soc Bras Med Trop 25: 275-276. Torres JRP (2009) Plasticidade neuronal mienterica no jejuno de ratos submetidos an infeccao aguda e cronica por cepa brasileira virulenta do tipo III de Toxoplasma gondii (Dissertation). Umuarama (PR), Universidade Paranaense. Odorizzi L (2009) Hipertrofia de neuronios mientericos nitrergicos de suinos com Toxoplasmose (Dissertation). Umuarama (PR), Universidade Paranaense. Soares J, Moreira NM, da Silva AV, Sant'Ana Dde M, Araújo EJ (2009) [Chronic infection due to Toxoplasma gondii inducing neuron hypertrophy of the myenteric plexus of Rattus norvegicus descending colon]. Rev Bras Parasitol Vet 18: 57-60. Rachinel N, Buzoni-Gatel D, Dutta C, Mennechet FJ, Luangsay S, et al. (2004) The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii. J Immunol 173: 2725-2735. Potasman I, Araujo FG, Desmonts G, Remington JS (1986) Analysis of Toxoplasma gondii antigens recognized by human sera obtained before and after acute infection. J Infect Dis 154: 650-657. Decoster A, Darcy F, Caron A, Capron A (1988) IgA antibodies against P30 as markers of congenital and acute toxoplasmosis. Lancet 2: 1104-1107. Khan IA, Eckel ME, Pfefferkorn ER, Kasper LH (1988) Production of gamma interferon by cultured human lymphocytes stimulated with a purified membrane protein (P30) from Toxoplasma gondii. J Infect Dis 157: 979-984. Neurath MF, Weigmann B, Finotto S, Glickman J, Nieuwenhuis E, et al. (2002) The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease. J Exp Med 195: 1129-1143. Guy-Grand D, DiSanto JP, Henchoz P, Malassis-Séris M, Vassalli P (1998) Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal. Eur J Immunol 28: 730-744. Mackay F, Browning JL, Lawton P, Shah SA, Comiskey M, et al. (1998) Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis. Gastroenterology 115: 1464-1475. Neurath MF, Pettersson S (1997) Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation. Immunobiology 198: 91-98. Kim K (2004) Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Trop 91: 69-81. Dou Z, Carruthers VB (2011) Cathepsin proteases in Toxoplasma gondii. Adv Exp Med Biol 712: 49-61. Chardès T, Velge-Roussel F, Mevelec P, Mevelec MN, Buzoni-Gatel D, et al. (1993) Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice. Immunology 78: 421-429. Else KJ, Grencis RK (1991) Cellular immune responses to the murine nematode parasite Trichuris muris. I. Differential cytokine production during acute or chronic infection. Immunology 72: 508-513. Grencis RK, Hültner L, Else KJ (1991) Host protective immunity to Trichinella spiralis in mice: activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74: 329-332. Solano Aguilar GI, Beshah E, Vengroski KG, Zarlenga D, Jauregui L, et al. (2001) Cytokine and lymphocyte profiles in miniature swine after oral infection with Toxoplasma gondii oocysts. Int J Parasitol 31: 187-195. Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, et al. (2011) Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats. Auton Neurosci 160: 3-8. Borutaité V, Brown GC (1996) Rapid reduction of nitric oxide by mitochondria, and reversible inhibition of mitochondrial respiration by nitric oxide. Biochem J 315 : 295-299. Dubey JP, Lindsay DS, Speer CA (1998) Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin Microbiol Rev 11: 267-299. Gopal R, Birdsell D, Monroy FP (2011) Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection. Parasite Immunol 33: 12-24. Mennechet FJ, Kasper LH, Rachinel N, Li W, Vandewalle A, et al. (2002) Lamina propria CD4+ T lymphocytes synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J Immunol 168: 2988-2996. Kasper L, Courret N, Darche S, Luangsay S, Mennechet F, et al. (2004) Toxoplasma gondii and mucosal immunity. Int J Parasitol 34: 401-409. Heimesaat MM, Fischer A, Jahn HK, Niebergall J, Freudenberg M, et al. (2007) Exacerbation of murine ileitis by Toll-like receptor 4 mediated sensing of lipopolysaccharide from commensal Escherichia coli. Gut 56: 941-948. Arciszewski M, Pierzynowski S, Ekblad E (2005) Lipopolysaccharide induces cell death in cultured porcine myenteric neurons. Dig Dis Sci 50: 1661-1668. Kobayashi M, Kweon MN, Kuwata H, Schreiber RD, Kiyono H, et al. (2003) Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice. J Clin Invest 111: 1297-1308. Cullen JJ, Caropreso DK, Ephgrave KS (1995) Effect of endotoxin on canine gastrointestinal motility and transit. J Surg Res 58: 90-95. De Winter BY, De Man JG, Seerden TC, Depoortere I, Herman AG, et al. (2004) Effect of ghrelin and growth hormone-releasing peptide 6 on septic ileus in mice. Neurogastroenterol Motil 16: 439-446. Cullen JJ, Caropreso DK, Ephgrave KS, Hemann LL, Hinkhouse MM (1997) The effect of endotoxin on canine jejunal motility and transit. J Surg Res 67: 54-57. Fan YP, Chakder S, Gao F, Rattan S (2001) Inducible and neuronal nitric oxide synthase involvement in lipopolysaccharide-induced sphincteric dysfunction. Am J Physiol Gastrointest Liver Physiol 280: G32-42. Strober W, Fuss I, Mannon P (2007) The fundamental basis of inflammatory bowel disease. J Clin Invest 117: 514-521. Liew FY (1993) The role of nitric oxide in parasitic diseases. Ann Trop Med Parasitol 87: 637-642. Ramanathan M, Giladi A, Leibovich SJ (2003) Regulation of vascular endothelial growth factor gene expression in murine macrophages by nitric oxide and hypoxia. Exp Biol Med (Maywood) 228: 697-705. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 157: 87-94. Brunet LR (2001) Nitric oxide in parasitic infections. Int Immunopharmacol 1: 1457-1467. Brown GC (1999) Nitric oxide and mitochondrial respiration. Biochim Biophys Acta 1411: 351-369. Brown GC, Borutaite V (1999) Nitric oxide, cytochrome c and mitochondria. Biochem Soc Symp 66: 17-25. Brown GC (2001) Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. Biochim Biophys Acta 1504: 46-57. Hindley S, Juurlink BH, Gysbers JW, Middlemiss PJ, Herman MA, et al. (1997) Nitric oxide donors enhance neurotrophin-induced neurite outgrowth through a cGMP-dependent mechanism. J Neurosci Res 47: 427-439. Truman JW, De Vente J, Ball EE (1996) Nitric oxide-sensitive guanylate cyclase activity is associated with the maturational phase of neuronal development in insects. Development 122: 3949-3958. Lonart G, Wang J, Johnson KM (1992) Nitric oxide induces neurotransmitter release from hippocampal slices. Eur J Pharmacol 220: 271-272. Hölscher C, Rose SP (1992) An inhibitor of nitric oxide synthesis prevents memory formation in the chick. Neurosci Lett 145: 165-167. Myslivecek J, Hassmannová J, Barcal J, Safanda J, Zalud V (1996) Inhibitory learning and memory in newborn rats influenced by nitric oxide. Neuroscience 71: 299-312. Wong JM, Billiar TR (1995) Regulation and function of inducible nitric oxide synthase during sepsis and acute inflammation. Adv Pharmacol 34: 155-170. Li J, Baud O, Vartanian T, Volpe JJ, Rosenberg PA (2005) Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci U S A 102: 9936-9941. Ridnour LA, Windhausen AN, Isenberg JS, Yeung N, Thomas DD, et al. (2007) Nitric oxide regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-dependent and -independent pathways. Proc Natl Acad Sci U S A 104: 16898-16903. Cirillo C, Sarnelli G, Esposito G, Grosso M, Petruzzelli R, et al. (2009) Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein. Neurogastroenterol Motil 21: 1209-1209e112. Cirillo C, Sarnelli G, Turco F, Mango A, Grosso M, et al. (2011) Proinflammatory stimuli activates human-derived enteroglial cells and induces autocrine nitric oxide production. Neurogastroenterol Motil 23: e372-382. Hokari R, Miura S, Fujimori H, Tsuzuki Y, Shigematsu T, et al. (1998) Nitric oxide modulates T-lymphocyte migration in Peyer's patches and villous submucosa of rat small intestine. Gastroenterology 115: 618-627. Ashwood P, Anthony A, Pellicer AA, Torrente F, Walker-Smith JA, et al. (2003) Intestinal lymphocyte populations in children with regressive autism: evidence for extensive mucosal immunopathology. J Clin Immunol 23: 504-517. Dubey JP (1997) Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii. J Eukaryot Microbiol 44: 592-602. Prgomet C, Prenner ML, Schwarz FJ, Pfaffl MW (2007) Effect of lactoferrin on selected immune system parameters and the gastrointestinal morphology in growing calves. J Anim Physiol Anim Nutr (Berl) 91: 109-119. Bogers J, Moreels T, De Man J, Vrolix G, Jacobs W, et al. (2000) Schistosoma mansoni infection causing diffuse enteric inflammation and damage of the enteric nervous system in the mouse small intestine. Neurogastroenterol Motil 12: 431-440. Balemba OB, Semuguruka WD, Hay-Schmidt A, Johansen MV, Dantzer V (2001) Vasoactive intestinal peptide and substance P-like immunoreactivities in the neteric nervous system of the pig correlate with the severity of pathological changes induced by Schisostoma japonicum. Int J Parasitol 31: 1503-1514. Bauer AJ (2008) Mentation on the immunological modulation of gastrointestinal motility. Neurogastroenterol Motil 20 Suppl 1: 81-90. Barbara G, Giorgio R (2004) Inflammation. Spiller R, Grundy D (eds), Pathophysiology of the Enteric Nervous System: A Basis for Understanding Functional Diseases. Blackwell Publishing, Oxford 61-78. S Ferreira GL, Mineo JR, Oliveira JG, V Ferro EA, Souza MA, et al. (2004) Toxoplasma gondii and mast cell interactions in vivo and in vitro: experimental infection approaches in Calomys callosus (Rodentia, Cricetidae). Microbes Infect 6: 172-181. Sand E, Themner-Persson A, Ekblad E (2009) Mast cells reduce survival of myenteric neurons in culture. Neuropharmacology 56: 522-530. Sang Q, Young HM (1996) Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse. Cell Tissue Res 284: 39-53. Cowen T, Johnson RJ, Soubeyre V, Santer RM (2000) Restricted diet rescues rat enteric motor neurones from age related cell death. Gut 47: 653-660. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, et al. (2003) Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421: 384-388. Perry EK, Lee ML, Martin-Ruiz CM, Court JA, Volsen SG, et al. (2001) Cholinergic activity in autism: abnormalities in the cerebral cortex and basal forebrain. Am J Psychiatry 158: 1058-1066. Chauhan A, Chauhan V (2006) Oxidative stress in autism. Pathophysiology 13: 171-181. Hardan AY, Handen BL (2002) A retrospective open trial of adjunctive donepezil in children and adolescents with autistic disorder. J Child Adolesc Psychopharmacol 12: 237-241. Gallowitsch-Puerta M, Pavlov VA (2007) Neuro-immune interactions via the cholinergic anti-inflammatory pathway. Life Sci 80: 2325-2329. Pavlov VA (2008) Cholinergic modulation of inflammation. Int J Clin Exp Med 1: 203-212. Jyonouchi H (2010) Autism spetrum disorders and allergy: observation from a pediatric allergy/immunology clinic. Expert Rev Clin Immunol 6: 397-411. Theoharides TC, Angelidou A, Alysandratos KD, Zhang B, Asadi S, et al. (2011) Mast cell activation and autism. Biochim Biophys Acta 1822: 34-41. Theoharides TC (2009) Autism spectrum disorders and mastocytosis. Int J Immunopathol Pharmacol 22: 859-865. Kilic G, Guler N, Suleyman A, Tamay Z (2010) Chronic urticaria and autoimmunity in children. Pediatr Allergy Immunol 21: 837-842. Croen LA, Grether JK, Yoshida CK, Odouli R, Van de Water J (2005) Maternal autoimmune diseases, asthma and allergies, and childhood autism spectrum disorders: a case-control study. Arch Pediatr Adolesc Med 159: 151-157. Sacco R, Curatolo P, Manzi B, Militerni R, Bravaccio C, et al. (2010) Principal pathogenetic components and biological endophenotypes in autism spectrum disorders. Autism Res 3: 237-252. Reed DE, Barajas-Lopez C, Cottrell G, Velazquez-Rocha S, Dery O, et al. (2003) Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons. J Physiol 547: 531-542. He S, Aslam A, Gaça MD, He Y, Buckley MG, et al. (2004) Inhibitors of tryptase as mast cell-stabilizing agents in the human airways: effects of tryptase and other agonists of proteinase-activated receptor 2 on histamine release. J Pharmacol Exp Ther 309: 119-126. Farhadi A, Fields JZ, Keshavarzian A (2007) Mucosal mast cells are pivotal elements in inflammatory bowel disease that connect the dots: stress, intestinal hyperpermeability and inflammation. World J Gastroenterol 13: 3027-3030. Kempuraj D, Asadi S, Zhang B, Manola A, Hogan J, et al. (2010) Mercury induces inflammatory mediator release from human mast cells. J Neuroinflammation 7: 20. Delong G (2011) A positive association found between autism prevalence and childhood vaccination uptake across the U.S. population. J Toxicol Environ Health A 74: 903-916. Bonapaz RS (2009) Efeitos do infeccao por oocistos de Toxoplasma gondii sobre a parede intestinal e o plexo mienterico de Gallus gallus (Dissertation). Umuarama (PR), Universidade Paranaense. Shiraishi CS, De Azevedo JF, Da Silva AV, Sant’Ana DMG, Araujo EJA (2009) Analise morfometrica de parede intestinal e dinamica de mucinas secretadas no ileo de frangos infectados por Toxoplasma gondii. Cienia Rural 39: 2146-2153. Dubey JP, Speer CA, Shen SK, Kwok OC, Blixt JA (1997) Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts. J Parasitol 83: 870-882. S Ferreira GL, Mineo JR, Oliveira JG, V Ferro EA, Souza MA, et al. (2004) Toxoplasma gondii and mast cell interactions in vivo and in vitro: experimental infection approaches in Calomys callosus (Rodentia, Cricetidae). Microbes Infect 6: 172-181. Gil CD, Mineo JR, Smith RL, Oliani SM (2002) Mast cells in the eyes of Calomys callosus(Rodentia: Cricetidae) infected by Toxoplasma gondii. Parasitol Res 88: 557-562. Henderson WR Jr, Chi EY (1998) The importance of leukotrienes in mast cell-mediated Toxoplasma gondii cytotoxicity. J Infect Dis 177: 1437-1443. Channon JY, Seguin RM, Kasper LH (2000) Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes. Infect Immun 68: 4822-4826. Nakao M, Konishi E (1991) Proliferation of Toxoplasma gondii in human neutrophils in vitro. Parasitology 103 Pt 1: 23-27. Wilson CB, Remington JS (1979) Activity of human blood leukocytes against Toxoplasma gondii. J Infect Dis 140: 890-895. Delemarre FG, Stevenhagen A, Kroon FP, van Eer MY, Meenhorst PL, et al. (1994) Effect of IFN-gamma on the proliferation of Toxoplasma gondii in monocytes and monocyte-derived macrophages from AIDS patients. Immunology 83: 646-650. McLeod R, Bensch KG, Smith SM, Remington JS (1980) Effects of human peripheral blood monocytes, monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii. Cell Immunol 54: 330-350. Murray HW, Rubin BY, Carriero SM, Harris AM, Jaffee EA (1985) Human mononuclear phagocyte antiprotozoal mechanisms: oxygen-dependent vs oxygen-independent activity against intracellular Toxoplasma gondii. J Immunol 134: 1982-1988. Murray HW, Szuro-Sudol A, Wellner D, Oca MJ, Granger AM, et al. (1989) Role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages. Infect Immun 57: 845-849. Fadul CE, Channon JY, Kasper LH (1995) Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes. Infect Immun 63: 4290-4294. Wilson CB, Westall J (1985) Activation of neonatal and adult human macrophages by alpha, beta, and gamma interferons. Infect Immun 49: 351-356. Anderson SE, Bautista S, Remington JS (1976) Induction of resistance to Toxoplasma gondii in human macrophages by soluble lymphocyte products. J Immunol 117: 381-387. Halonen SK, Lyman WD, Chiu FC (1996) Growth and development of Toxoplasma gondii in human neurons and astrocytes. J Neuropathol Exp Neurol 55: 1150-1156. Pfefferkorn ER (1984) Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc Natl Acad Sci U S A 81: 908-912. Pfefferkorn ER, Guyre PM (1984) Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon. Infect Immun 44: 211-216. Woodman JP, Dimier IH, Bout DT (1991) Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replication. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. J Immunol 147: 2019-2023. Nagineni CN, Pardhasaradhi K, Martins MC, Detrick B, Hooks JJ (1996) Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells. Infect Immun 64: 4188-4196. Peterson PK, Gekker G, Hu S, Chao CC (1995) Human astrocytes inhibit intracellular multiplication of Toxoplasma gondii by a nitric oxide-mediated mechanism. J Infect Dis 171: 516-518. Chao CC, Gekker G, Hu S, Peterson PK (1994) Human microglial cell defense against Toxoplasma gondii. The role of cytokines. J Immunol 152: 1246-1252. Theoharides TC, Kalogeromitros D (2006) The critical role of mast cells in allergy and inflammation. Ann N Y Acad Sci 1088: 78-99. Mayr SI, Zuberi RI, Liu FT (2003) Role of immunoglobulin E and mast cells in murine models of asthma. Braz J Med Biol Res 36: 821-827. Saeij JP, Boyle JP, Boothroyd JC (2005) Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol 21: 476-481. Dubey JP (1998) Advances in the life cycle of Toxoplasma gondii. Int J Parasitol 28: 1019-1024. Buzoni-Gatel D, Debbabi H, Mennechet FJ, Martin V, Lepage AC, et al. (2001) Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes. Gastroenterology 120: 914-924. Lidar M, Langevitz P, Shoenfeld Y (2009) The role of infection in inflammatory bowel disease: initiation, exacerbation and protection. Isr Med Assoc J 11: 558-563. Plot L, Amital H, Barzilai O, Ram M, Nicola B, et al. (2009) Infections may have a protective role in the etiopathogenesis of celiac disease. Ann N Y Acad Sci 1173: 670-674. Egan CE, Cohen SB, Denkers EY (2012) Insights into inflammatory bowel disease using Toxoplasma gondii as an infectious trigger. Immunol Cell Biol 90: 668-675. Hertervig E, Wieslander J, Johansson C, Wiik A, Nilsson A (1995) Anti-neutrophil cytoplasmic antibodies in chronic inflammatory bowel disease. Prevalence and diagnostic role. Scand J Gastroenterol 30: 693-698. Proujansky R, Fawcett PT, Gibney KM, Treem WR, Hyams JS (1993) Examination of anti-neutrophil cytoplasmic antibodies in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 17: 193-197. Seibold F, Weber P, Klein R, Berg PA, Wiedmann KH (1992) Clinical significance of antibodies against neutrophils in patients with inflammatory bowel disease and primary sclerosing cholangitis. Gut 33: 657-662. Colombel JF, Reumaux D, Duthilleul P, Noël LH, Gower-Rousseau C, et al. (1992) Antineutrophil cytoplasmic autoantibodies in inflammatory bowel diseases. Gastroenterol Clin Biol 16: 656-660. Lombardi G, Annese V, Piepoli A, Bovio P, Latiano A, et al. (2000) Antineutrophil cytoplasmic antibodies in inflammatory bowel disease: clinical role and review of the literature. Dis Colon Rectum 43: 999-1007. Hinze-Selch D, Däubener W, Erdag S, Wilms S (2010) The diagnosis of a personality disorder increases the likelihood for seropositivity to Toxoplasma gondii in psychiatric patients. Folia Parasitol (Praha) 57: 129-135. Alvarado-Esquivel C, Estrada-Martínez S (2011) Toxoplasma gondii infection and abdominal hernia: evidence of a new association. Parasit Vectors 4: 112. Del Rio L, Bennouna S, Salinas J, Denkers EY (2001) CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection. J Immunol 167: 6503-6509. Hack M, Horbar JD, Malloy MH, Tyson JE, Wright E, et al. (1991) Very low birth weight outcomes of the National Institute of Child Health and Human Development Neonatal Network. Pediatrics 87: 587-597. Uauy RD, Fanaroff AA, Korones SB, Phillips EA, Phillips JB, et al. (1991) Necrotizing enterocolitis in very low birth weight infants: biodemographic and clinical correlates. National Institute of Child Health and Human Development Neonatal Network. J Pediatr 119: 630-638. MacKendrick W, Caplan M (1993) Necrotizing enterocolitis. New thoughts about pathogenesis and potential treatments. Pediatr Clin North Am 40: 1047-1059. Giannone PJ, Luce WA, Nankervis CA, Hoffman TM, Wold LE (2008) Necrotizing enterocolitis in neonates with congenital heart disease. Life Sci 82: 341-347. Kosloske AM (1994) Epidemiology of necrotizing enterocolitis. Acta Paediatr Suppl 396: 2-7. Ulevitch RJ, Tobias PS (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 13: 437-457. Thompson AM, Bizzarro MJ (2008) Necrotizing enterocolitis in newborns: pathogenesis, prevention and management. Drugs 68: 1227-1238. Nanthakumar NN, Fusunyan RD, Sanderson I, Walker WA (2000) Inflammation in the developing human intestine: A possible pathophysiologic contribution to necrotizing enterocolitis. Proc Natl Acad Sci U S A 97: 6043-6048. Fusunyan RD, Nanthakumar NN, Baldeon ME, Walker WA (2001) Evidence for an innate immune response in the immature human intestine: toll-like receptors on fetal enterocytes. Pediatr Res 49: 589-593. Jilling T, Simon D, Lu J, Meng FJ, Li D, et al. (2006) The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis. J Immunol 177: 3273-3282. Spear W, Chan D, Coppens I, Johnson RS, Giaccia A, et al. (2006) The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels. Cell Microbiol 8: 339-352. Prandota J (2010) Migraine associated with patent foramen ovale may be caused by reactivation of cerebral toxoplasmosis triggered by arterial blood oxygen desaturation. Int J Neurosci 120: 81-87. Juul SE, Joyce AE, Zhao Y, Ledbetter DJ (1999) Why is erythropoietin present in human milk? Studies of erythropoietin receptors on enterocytes of human and rat neonates. Pediatr Res 46: 263-268. McPherson RJ, Juul SE (2007) High-dose erythropoietin inhibits apoptosis and stimulates proliferation in neonatal rat intestine. Growth Horm IGF Res 17: 424-430. Prandota J (2012) Rhesus-associated glycoprotein (RhAG) phenotype of the red blood cells modulates T. gondii infection-associated psychomotor performance reaction times and changes in the human personality profile. Impaired function of the CO2, AGP1, and AQP4 gas channels may cause hypoxia and thus enhance neuroinflammation in autistic individuals. Gemma C (ed), Neuroinflammation: Pathogenesis, Mechanisms and Management. Nova Publishers, New York. Olariu TR, Remington JS, McLeod R, Alam A, Montoya JG (2011) Severe congenital toxoplasmosis in the United States: clinical and serologic findings in untreated infants. Pediatr Infect Dis J 30: 1056-1061. Ajzenberg D, Cogne N, Paris L, Bessieres MH, Thulliez P, et al. (2002) Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J Infect Dis 186: 684-689. Hide G, Morley EK, Hughes JM, Gerwash O, Elmahaishi MS, et al. (2009) Evidence for high levels of vertical transmission in Toxoplasma gondii. Parasitology 136: 1877-1885. Durandy A (2001) [Development of specific immunity in prenatal life]. Arch Pediatr 8: 979-985. Lindsay DS, Dubey JP (2011) Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis. Parasitology . Sigge W, Wedel T, Kühnel W, Krammer HJ (1998) Morphologic alterations of the enteric nervous system and deficiency of non-adrenergic non-cholinergic inhibitory innervation in neonatal necrotizing enterocolitis. Eur J Pediatr Surg 8: 87-94. Wedel T, Krammer HJ, Kühnel W, Sigge W (1998) Alterations of the enteric nervous system in neonatal necrotizing enterocolitis revealed by whole-mount immunohistochemistry. Pediatr Pathol Lab Med 18: 57-70. Bush TG (2002) Enteric glial cells. An upstream target for induction of necrotizing enterocolitis and Crohn's disease? Bioessays 24: 130-140. Bailey A, Luthert P, Dean A, Harding B, Janota I, et al. (1998) A clinicopathological study of autism. Brain 121 : 889-905. Fatemi SH, Halt AR, Realmuto G, Earle J, Kist DA, et al. (2002) Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol 22: 171-175. Arndt TL, Stodgell CJ, Rodier PM (2005) The teratology of autism. Int J Dev Neurosci 23: 189-199. Ingram JL, Peckham SM, Tisdale B, Rodier PM (2000) Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism. Neurotoxicol Teratol 22: 319-324. Zeng K, Wang X, Xi Z, Yan Y (2010) Adverse effects of carbamazepine, phenytoin, valproate and lamotrigine monotherapy in epileptic adult Chinese patients. Clin Neurol Neurosurg 112: 291-295. Jahromi SR, Togha M, Fesharaki SH, Najafi M, Moghadam NB, et al. (2011) Gastrointestinal adverse effects of antiepileptic drugs in intractable epileptic patients. Seizure 20: 343-346. Baig MA, Rasheed J, Lin YS, Smith P (2006) Necrotizing enterocolitis with hepatic portal venous gas and pneumatosis intestinalis in a patient with AIDS. Internet J Infect Dis 5. Pear BL (1998) Pneumatosis intestinalis: a review. Radiology 207: 13-19. Zhu X, Fan WG, Li DP, Kung H, Lin MC (2011) Heme oxygenase-1 system and gastrointestinal inflammation: a short review. World J Gastroenterol 17: 4283-4288. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, et al. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 6: 422-428. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 103: 129-135. Hamprecht K, Maschmann J, Vochem M, Dietz K, Speer CP, et al. (2001) Epidemiology of transmission of cytomegalovirus from mother to preterm infant by breastfeeding. Lancet 357: 513-518. Gessler P, Bischoff GA, Wiegand D, Essers B, Bossart W (2004) Cytomegalovirus-associated necrotizing enterocolitis in a preterm twin after breastfeeding. J Perinatol 24: 124-126. Sann L, Aymard M, Gibert R, Bourgeois J, Cottancin G, et al. (1981) [Necrotizing enterocolitis and cytomegalovirus infection (author's transl)]. Nouv Presse Med 10: 2495-2499. D'Agostino S, Stracca-Pansa V, Drei F, Valli F, Colombo B, et al. (1988) [Post-necrotizing enterocolitis stenosis of the colon associated with cytomegalovirus infection. Description of a clinical case]. Pediatr Med Chir 10: 637-639. Reyes C, Pereira S, Warden MJ, Sills J (1997) Cytomegalovirus enteritis in a premature infant. J Pediatr Surg 32: 1545-1547. Srinivasjois RM, Kava MP, Thomas A, Rao SC (2008) Cytomegalovirus-associated ileal stricture in a preterm neonate. J Paediatr Child Health 44: 80-82. Huang YC, Lin TY, Huang CS, Hseun C (1996) Ileal perforation caused by congenital or perinatal cytomegalovirus infection. J Pediatr 129: 931-934. Bang S, Park YB, Kang BS, Park MC, Hwang MH, et al. (2004) CMV enteritis causing ileal perforation in underlying lupus enteritis. Clin Rheumatol 23: 69-72. Cha JM, Lee JI, Choe JW, Joo KR, Jung SW, et al. (2010) Cytomegalovirus enteritis causing ileal perforation in an elderly immunocompetent individual. Yonsei Med J 51: 279-283. Almeida N, Romãozinho JM, Amaro P, Ferreira M, Cipriano MA, et al. (2009) Fatal mid-gastrointestinal bleeding by cytomegalovirus enteritis in an immunocompetent patient. Acta Gastroenterol Belg 72: 245-248. Choi SW, Chung JP, Song YK, Park YN, Chu JK, et al. (2001) Lower gastrointestinal bleeding due to cytomegalovirus ileal ulcers in an immunocompetent man. Yonsei Med J 42: 147-151. Lin WR, Su MY, Hsu CM, Ho YP, Ngan KW, et al. (2005) Clinical and endoscopic features for alimentary tract cytomegalovirus disease: report of 20 cases with gastrointestinal cytomegalovirus disease. Chang Gung Med J 28: 476-484. Rafailidis PI, Mourtzoukou EG, Varbobitis IC, Falagas ME (2008) Severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review. Virol J 5: 47. Tamer GS, Dundar D, Caliskan E (2009) Seroprevalence of Toxoplasma gondii, rubella and cytomegalovirus among pregnant women in western region of Turkey. Clin Invest Med 32: E43-47. Inagaki AD, Oliveira LA, Oliveira MF, Santos RC, Araújo RM, et al. (2009) [Seroprevalence of antibodies for toxoplasmosis, rubella, cytomegalovirus, syphilis and HIV among pregnant women in Sergipe]. Rev Soc Bras Med Trop 42: 532-536. Ocak S, Zeteroglu S, Ozer C, Dolapcioglu K, Gungoren A (2007) Seroprevalence of Toxoplasma gondii, rubella and cytomegalovirus among pregnant women in southern Turkey. Scand J Infect Dis 39: 231-234. Shakhgil'dian VI, Vasil'eva TE, Peregudova AB, Gruzdev BM, Danilova TV, et al. (2008) [Spectrum, clinical features, diagnosis of opportunistic and comorbid pathology in HIV-infected patients admitted to infection hospital of Moscow]. Ter Arkh 80: 10-17. Afzal N, Murch S, Thirrupathy K, Berger L, Fagbemi A, et al. (2003) Constipation with acquired megarectum in children with autism. Pediatrics 112: 939-942. Wedel T, Roblick U, Gleiss J, Ott V, Eggers R, et al. (1999) [Disorders of intestinal innervation as a possible cause for chronic constipation]. Zentralbl Chir 124: 796-803. Wedel T, Gleiss J, Schiedeck T, Herold A, Bruch HP (1998) [Megacolon in adults--the spectrum of underlying intestinal innervation disorders]. Langenbecks Arch Chir Suppl Kongressbd 115: 979-981. Tomita R, Munakata K, Howard ER, Fujisaki S (2004) Histological studies on Hirschsprung’s disease and its allied disorders in childhood. Hepatogastroenterology 51: 1042-1044. Wedel T, Böttner M, Krammer HJ (2007) [The enteric nervous system and interstitial cells of Cajal. Changes in chronic constipation in adults]. Pathologe 28: 143-148. Wester T, O'Briain DS, Puri P (1999) Notable postnatal alterations in the myenteric plexus of normal human bowel. Gut 44: 666-674. Meier-Ruge W (1971) [Casuistic of colon disorder with symptoms of Hirschsprung's disease (author's transl)]. Verh Dtsch Ges Pathol 55: 506-510. Gittes GK, Kim J, Yu G, de Lorimier AA (1993) Severe constipation with diffuse intestinal myenteric hyperganglionosis. J Pediatr Surg 28: 1630-1632. Zaniolo LM, da Silva AV, Sant'Ana Dde M, Araújo EJ (2012) Toxoplasma gondii infection causes morphological changes in caecal myenteric neurons. Exp Parasitol 130: 103-109. Yaman K, Akarsu GA, Güngör Ç, AtaoÄŸlu H (2011) [Characterization of Toxoplasma gondii proteins from various strains]. Turkiye Parazitol Derg 35: 133-136. Watson WC, Sullivan SN, Corke M, Rush D (1978) Globus and headache: common symptoms of the irritable bowel syndrome. Can Med Assoc J 118: 387-388. Jones R, Lydeard S (1992) Irritable bowel syndrome in the general population. BMJ 304: 87-90. Mavromichalis I, Zaramboukas T, Giala MM (1995) Migraine of gastrointestinal origin. Eur J Pediatr 154: 406-410. Mulak A, Paradowski L (2005) [Migraine and irritable bowel syndrome]. Neurol Neurochir Pol 39: S55-60. Whitehead WE, Palsson OS, Levy RR, Feld AD, Turner M, et al. (2007) Comorbidity in irritable bowel syndrome. Am J Gastroenterol 102: 2767-2776. Brams W (1922) Abdominal migraine. JAMA 78: 26-27. Popovich DM, Schentrup DM, McAlhany AL (2010) Recognizing and diagnosing abdominal migraines. J Pediatr Health Care 24: 372-377. Carson L, Lewis D, Tsou M, McGuire E, Surran B, et al. (2011) Abdominal migraine: an under-diagnosed cause of recurrent abdominal pain in children. Headache 51: 707-712. Mortimer MJ, Kay J, Jaron A (1993) Clinical epidemiology of childhood abdominal migraine in an urban general practice. Dev Med Child Neurol 35: 243-248. Dignan F, Abu-Arafeh I, Russell G (2001) The prognosis of childhood abdominal migraine. Arch Dis Child 84: 415-418. Lewis DW (2009) Pediatric migraine. Neurol Clin 27: 481-501. Battistella PA, Fiumana E, Binelli M, Bertossi E, Battista P, et al. (2006) Primary headaches in preschool age children: clinical study and follow-up in 163 patients. Cephalalgia 26: 162-171. Battistella PA, Toldo I (2006) Headache and recurrent abdominal pains in preschool children. J Headache Pain 7: 322-323. Farmachidi JP, Sobesky R, Coffin B (2002) Prevalence des troubles fonctionelles digestifs chez des patients migraineux chroniques. Gastroenterol Clin 26: A399. Prandota J (2007) Recurrent headache as the main symptom of acquired cerebral toxoplasmosis in non-human immunodeficiency virus infected subjects with no lymphadenopathy. The parasite may be responsible for the neurogenic inflammation postulated as a cause of different types of headaches. Am J Ther 14: 63-105. Prandota J (2012) Idiopathic intracranial hypertension may be caused by reactivation of latent cerebral toxoplasmosis. Effect of various diseases and clinical states. Gemma C (ed), Neuroinflammation: Pathogenesis, Mechanisms and Management, Nova Publishers. Prandota J (2012) Idiopathic intracranial hypertension may be caused by reactivation of latent cerebral toxoplasmosis probably because of disturbances in the host and/or Toxoplasma gondii immune defense mechanisms. Effect of various medications and biologic agents. Gemma C (ed). Neuroinflammation: Pathogenesis, Mechanisms and Management, Nova Publishers. Russell G, Abu-Arafeh I, Symon DN (2002) Abdominal migraine: evidence for existence and treatment options. Paediatr Drugs 4: 1-8. Corbo J (2003) The role of anticonvulsants in preventive migraine therapy. Curr Pain Headache Rep 7: 63-66. Tan V, Sahami AR, Peebles R, Shaw RJ (2006) Abdominal migraine and treatment with intravenous valproic Acid. Psychosomatics 47: 353-355. Jones-Brando L, Torrey EF, Yolken R (2003) Drugs used in the treatment of schizophrenia and bipolar disorder inhibit the replication of Toxoplasma gondii. Schizophr Res 62: 237-244. Grundy SM (2006) Metabolic syndrome: connecting and reconciling cardiovascular and diabetes worlds. J Am Coll Cardiol 47: 1093-1100. Nagahori M, Hyun SB, Totsuka T, Okamoto R, Kuwahara E, et al. (2010) Prevalence of metabolic syndrome is comparable between inflammatory bowel disease patients and the general population. J Gastroenterol 45: 1008-1013. Yorulmaz E, Adali G, Yorulmaz H, Ulasoglu C, Tasan G, et al. (2011) Metabolic syndrome frequency in inflammatory bowel diseases. Saudi J Gastroenterol 17: 376-382. Kaser A, Blumberg RS (2009) Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease. Semin Immunol 21: 156-163. Kaser A, Blumberg RS (2010) Endoplasmic reticulum stress and intestinal inflammation. Mucosal Immunol 3: 11-16. Niederreiter L, Kaser A (2011) Endoplasmic reticulum stress and inflammatory bowel disease. Acta Gastroenterol Belg 74: 330-333. Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, et al. (2008) Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 5: e54 Kim YS, Ho SB (2010) Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep 12: 319-330. Mendoza JL, Abreu MT (2009) Biological markers in inflammatory bowel disease: practical consideration for clinicians. Gastroenterol Clin Biol 33 Suppl 3: S158-173. Xiong N, Ji C, Li Y, He Z, Bo H, et al. (2009) The physical status of children with autism in China. Res Dev Disabil 30: 70-76. Curtin C, Bandini LG, Perrin EC, Tybor DJ, Must A (2005) Prevalence of overweight in children and adolescents with attention deficit hyperactivity disorder and autism spectrum disorders: a chart review. BMC Pediatr 5: 48. Sugiyama T (1991) A research of obesity in autism. Jap J Dev Disabil 13: 53-58. Takeuchi E (1994) Incidence of obesity among school children with mental retardation in Japan. Am J Ment Retard 99: 283-288. Arsenijevic D, Girardier L, Seydoux J, Pechere JC, Garcia I, et al. (1998) Metabolic-cytokine responses to a second immunological challenge with LPS in mice with T. gondii infection. Am J Physiol 274: E439-445. Portugal LR, Fernandes LR, Pietra Pedroso VS, Santiago HC, Gazzinelli RT, et al. (2008) Influence of low-density lipoprotein (LDL) receptor on lipid composition, inflammation and parasitism during Toxoplasma gondii infection. Microbes Infect 10: 276-284. Aygun AD, Gungor S, Ustundag B, Gurgoze MK, Sen Y (2005) Proinflammatory cytokines and leptin are increased in serum of prepubertal obese children. Mediators Inflamm 2005: 180-183. GÅ‚owiÅ„ska B, Urban M (2003) [Selected cytokines (Il-6, Il-8, Il-10, MCP-1, TNF-alpha) in children and adolescents with atherosclerosis risk factors: obesity, hypertension, diabetes]. Wiad Lek 56: 109-116. Wiest MM, German JB, Harvey DJ, Watkins SM, Hertz-Picciotto I (2009) Plasma fatty acid profiles in autism: a case-control study. Prostaglandins Leukot Essent Fatty Acids 80: 221-227. Milovanović I, Vujanić M, Klun I, Bobić B, Nikolić A, et al. (2009) Toxoplasma gondii infection induces lipid metabolism alterations in the murine host. Mem Inst Oswaldo Cruz 104: 175-178. Charron AJ, Sibley LD (2002) Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J Cell Sci 115: 3049-3059. Coppens I (2006) Contribution of host lipids to Toxoplasma pathogenesis. Cell Microbiol 8: 1-9. Nelson MM, Jones AR, Carmen JC, Sinai AP, Burchmore R, et al. (2008) Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii. Infect Immun 76: 828-844. Gupta N, Zahn MM, Coppens I, Joiner KA, Voelker DR (2005) Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth. J Biol Chem 280: 16345-16353. Zhang J, Shi GP (2011) Mast cells and metabolic syndrome. Biochim Biophys Acta. Hibi T, Ogata H (2007) Metabolic syndrome and inflammatory bowel disease. Internal Medicine, Symposium III, 107-108. Butcher BA, Kim L, Johnson PF, Denkers EY (2001) Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B. J Immunol 167: 2193-2201. Suzuki Y (2002) Immunopathogenesis of cerebral toxoplasmosis. J Infect Dis 186 Suppl 2: S234-240. Jay M, Kojima S, Gillespie MN (1986) Nicotine potentiates superoxide anion generation by human neutrophils. Toxicol Appl Pharmacol 86: 484-487. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H, Verdu EF, Collins SM (2006) The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology 131: 1122-1130. Ghia JE, Blennerhassett P, El-Sharkawy RT, Collins SM (2007) The protective effect of the vagus nerve in a murine model of chronic relapsing colitis. Am J Physiol Gastrointest Liver Physiol 293: G711-718. Yoshikawa H, Kurokawa M, Ozaki N, Nara K, Arou K, et al. (2006) Nicotine inhibits the production of proinflammatory mediators in human monocytes by suppression of 1-kappaB phosphorylation and nuclear factor-kappaB transcriptional activity through nicotinic acetylchoiline receptor alpha7. Clin Exp Immunol 146: 116-123. Shrestha SP, Tomita T, Weiss LM, Orlofsky A (2006) Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell. Int J Parasitol 36: 433-441. Gillespie MN, Owasoyo JO, Kojima S, Jay M (1987) Enhanced chemotaxis and superoxide anion production by polymorphonuclear leukocytes from nicotine-treated and smoke-exposed rats. Toxicology 45: 45-52. Nussler AK, Di Silvio M, Billiar TR, Hoffman RA, Geller DA, et al. (1992) Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J Exp Med 176: 261-264. Boyer L, Sidpra D, Jevon G, Buchan AM, Jacobson K (2007) Differential responses of VIPergic and nitrergic neurons in paediatric patients with Crohn's disease. Auton Neurosci 134: 106-114. Brandt CT, Tam PK, Gould SJ (1996) Nitrergic innervation of the human gut during early fetal development. J Pediatr Surg 31: 661-664. Riordan AM, Ruxton CH, Hunter JO (1998) A review of associations between Crohn's disease and consumption of sugars. Eur J Clin Nutr 52: 229-238. Nelson MM, Jones AR, Carmen JC, Sinai AP, Burchmore R, et al. (2008) Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii. Infect Immun 76: 828-844. Ding Y, Lu B, Chen D, Meng L, Shen Y, et al. (2010) Proteomic analysis of colonic mucosa in a rat model of irritable bowel syndrome. Proteomics 10: 2620-2630. Ding Y, Lu B, Meng LN, Fan YH, Guo Y, et al. (2010) Proteomics expression analysis of colonic mucosa in a rat model of irritable bowel syndrome. Zhonghua Yi Xue Za Zhi 90: 564-569. Butterfield DA, Lange ML (2009) Multifunctional roles of enolase in Alzheimer's disease brain: beyond altered glucose metabolism. J Neurochem 111: 915-933. Marshall ES, Elshekiha HM, Hakimi MA, Flynn RJ (2011) Toxoplasma gondii peroxiredoxin promotes altered macrophage function, caspase-1-dependent IL-1β secretion enhances parasite replication. Vet Res 42: 80. Karpuzoglu-Sahin E, Zhi-Jun Y, Lengi A, Sriranganathan N, Ansar Ahmed S (2001) Effects of long-term estrogen treatment on IFN-gamma, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice. Cytokine 14: 208-217. Dai R, Phillips RA, Karpuzoglu E, Khan D, Ahmed SA (2009) Estrogen regulates transcription factors STAT-1 and NF-kappaB to promote inducible nitric oxide synthase and inflammatory responses. J Immunol 183: 6998-7005. Ahmed SA, Hissong BD, Verthelyi D, Donner K, Becker K, et al. (1999) Gender and risk of autoimmune diseases: possible role of estrogenic compounds. Environ Health Perspect 107 Suppl 5: 681-686. Szekeres-Bartho J, Barakonyi A, Par G, Polgar B, Palkovics T, et al. (2001) Progesterone as an immunomodulatory molecule. Int Immunopharmacol 1: 1037-1048. Raghupathy R, Al-Mutawa E, Al-Azemi M, Makhseed M, Azizieh F, et al. (2009) Progesterone-induced blocking factor (PIBF) modulates cytokine production by lymphocytes from women with recurrent miscarriage or preterm delivery. J Reprod Immunol 80: 91-99. Druckmann R, Druckmann MA (2005) Progesterone and the immunology of pregnancy. J Steroid Biochem Mol Biol 97: 389-396. Hudic I, Fatusic Z (2009) Progesterone-induced blocking factor (PIBF) and Th1/Th2 cytokine in women with threatened spontaneous abortion. J Perinat Med 37: 338-342. Ferguson DJ (2009) Identification of faecal transmission of Toxoplasma gondii: Small science, large characters. Int J Parasitol 39: 871-875. Yazar S, Eser B, Yay M (2006) Prevalence of anti-toxoplasma Gondii antibodies in Turkish blood donors. Ethiop Med J 44: 257-261. Elhence P, Agarwal P, Prasad KN, Chaudhary RK (2010) Seroprevalence of Toxoplasma gondii antibodies in North Indian blood donors: implications for transfusion transmissible toxoplasmosis. Transfus Apher Sci 43: 37-40. Silveira C, Vallochi AL, Rodrigues da Silva U, Muccioli C, Holland GN, et al. (2011) Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis. Br J Ophthalmol 95: 396-400. Hafid J, Bellete B, Flori P, Sawadogo P, Boyer Y, et al. (2005) Materno-foetal transmission of murine toxoplasmosis after oral infection. Am J Immunol 1: 1-5. Schröder J (1975) Transplacental passage of blood cells. J Med Genet 12: 230-242. Fischer SA (2006) Infections complicating solid organ transplantation. Surg Clin North Am 86: 1127-1145, v-vi. Edvinsson B, Lundquist J, Ljungman P, Ringdén O, Evengård B (2008) A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation. APMIS 116: 345-351. Fricker-Hidalgo H, Bulabois CE, Brenier-Pinchart MP, Hamidfar R, Garban F, et al. (2009) Diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of DNA detection and serological techniques. Clin Infect Dis 48: e9-9e15. Laibe S, Ranque S, Curtillet C, Faraut F, Dumon H, et al. (2006) Timely diagnosis of disseminated toxoplasmosis by sputum examination. J Clin Microbiol 44: 646-648. Hiramoto RM, Mayrbaurl-Borges M, Galisteo AJ Jr, Meireles LR, Macre MS, et al. (2001) Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese. Rev Saude Publica 35: 113-118. Camossi LG, Greca-Júnior H, Corrêa AP, Richini-Pereira VB, Silva RC, et al. (2011) Detection of Toxoplasma gondii DNA in the milk of naturally infected ewes. Vet Parasitol 177: 256-261. Arantes TP, Lopes WD, Ferreira RM, Pieroni JS, Pinto VM, et al. (2009) Toxoplasma gondii: Evidence for the transmission by semen in dogs. Exp Parasitol 123: 190-194. Joiner KA, Dubremetz JF (1993) Toxoplasma gondii: a protozoan for the nineties. Infect Immun 61: 1169-1172. Lambert H, Dellacasa-Lindberg I, Barragan A (2011) Migratory responses of leukocytes infected with Toxoplasma gondii. Microbes Infect 13: 96-102. Lambert H, Hitziger N, Dellacasa I, Svensson M, Barragan A (2006) Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination. Cell Microbiol 8: 1611-1623. Da Gama LM, Ribeiro-Gomes FL, Guimarães U Jr, Arnholdt AC (2004) Reduction in adhesiveness to extracellular matrix components, modulation of adhesion molecules and in vivo migration of murine macrophages infected with Toxoplasma gondii. Microbes Infect 6: 1287-1296. Courret N, Darche S, Sonigo P, Milon G, Buzoni-Gâtel D, et al. (2006) CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. Blood 107: 309-316. Lambert H, Vutova PP, Adams WC, Loré K, Barragan A (2009) The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect Immun 77: 1679-1688. Bierly AL, Shufesky WJ, Sukhumavasi W, Morelli AE, Denkers EY (2008) Dendritic cells expressing plasmacytoid marker PDCA-1 are Trojan horses during Toxoplasma gondii infection. J Immunol 181: 8485-8491. Persson CM, Lambert H, Vutova PP, Dellacasa-Lindberg I, Nederby J, et al. (2009) Transmission of Toxoplasma gondii from infected dendritic cells to natural killer cells. Infect Immun 77: 970-976. Lambert H, Barragan A (2010) Modelling parasite dissemination: host cell subversion and immune evasion by Toxoplasma gondii. Cell Microbiol 12: 292-300. Persson EK, Agnarson AM, Lambert H, Hitziger N, Yagita H, et al. (2007) Death receptor ligation or exposure to perforin trigger rapid egress of the intracellular parasite Toxoplasma gondii. J Immunol 179: 8357-8365. Stray-Pedersen B (1993) Toxoplasmosis in pregnancy. Baillieres Clin Obstet Gynaecol 7: 107-137. Tenter AM, Heckeroth AR, Weiss LM (2000) Toxoplasma gondii: from animals to humans. Int J Parasitol 30: 1217-1258. Vidigal PV, Santos DV, Castro FC, Couto JC, Vitor RW, et al. (2002) Prenatal toxoplasmosis diagnosis from amniotic fluid by PCR. Rev Soc Bras Med Trop 35: 1-6. Sever JL, Ellenberg JH, Ley AC, Madden DL, Fuccillo DA, et al. (1988) Toxoplasmosis: maternal and pediatric findings in 23,000 pregnancies. Pediatrics 82: 181-192. Abbasi M, Kowalewska-Grochowska K, Bahar MA, Kilani RT, Winkler-Lowen B, et al. (2003) Infection of placental trophoblasts by Toxoplasma gondii. J Infect Dis 188: 608-616. El-Haddad MA, Desai M, Gayle D, Ross MG (2004) In utero development of fetal thirst and appetite: potential for programming. J Soc Gynecol Investig 11: 123-130. Harman CR (2008) Amniotic fluid abnormalities. Semin Perinatol 32: 288-294. Lo ES, Lo YM, Hjelm NM, Thilaganathan B (1998) Transfer of nucleated maternal cells into fetal circulation during the second trimester of pregnancy. Br J Haematol 100: 605-606. Cuddapah Sunku C, Gadi VK, de Laval de Lacoste B, Guthrie KA, Nelson JL (2010) Maternal and fetal microchimerism in granulocytes. Chimerism 1. Jonsson AM, Uzunel M, Götherström C, Papadogiannakis N, Westgren M (2008) Maternal microchimerism in human fetal tissues. Am J Obstet Gynecol 198: 325. Fuksman RB, Mazzitelli NG (2009) Second-trimester histopathological placental findings in maternal-fetal inflammatory response syndrome. Pediatr Dev Pathol 12: 42-46. Maloney S, Smith A, Furst DE, Myerson D, Rupert K, et al. (1999) Microchimerism of maternal origin persists into adult life. J Clin Invest 104: 41-47. Boyer K, Hill D, Mui E, Wroblewski K, Karrison T, et al. (2011) Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin Infect Dis 53: 1081-1089. Howell S, Talley NJ, Quine S, Poulton R (2004) The irritable bowel syndrome has origins in the childhood socioeconomic environment. Am J Gastroenterol 99: 1572-1578. Bengtson MB, Rønning T, Vatn MH, Harris JR (2006) Irritable bowel syndrome in twins: genes and environment. Gut 55: 1754-1759. de Boissieu D, Bargaoui K, Sakiroglu O, Francoual C, Dupont C, et al. (1989) [Esophagogastroduodenitis in the newborn. Apropos of 32 cases]. Arch Fr Pediatr 46: 711-715. de Boissieu D, Dupont C, Barbet JP, Bargaoui K, Badoual J (1994) Distinct features of upper gastrointestinal endoscopy in the newborn. J Pediatr Gastroenterol Nutr 18: 334-338. Boukthir S, Fetni I, M'Rad S, Bennour F, Barsaoui S (2002) [Esophagogastroduodenitis in the newborn. Report of 90 cases]. Tunis Med 80: 18-20. BERANBAUM SL, WALDRON RJ (1952) Chronic ulcerative colitis; case report in a newborn infant. Pediatrics 9: 773-778. Alekseevskikh IuG, Tagiev NA (1974) [Ulcerative-necrotic disease of the intestine in premature and newborn infants (literature survey)]. Vopr Okhr Materin Det 19: 59-63. Vanderborght M, Nassogne MC, Hermans D, Moniotte S, Seneca S, et al. (2004) Intractable ulcerative colitis of infancy in a child with mitochondrial respiratory chain disorder. J Pediatr Gastroenterol Nutr 38: 355-357. Zhang H, Li GN, Liu XH (2009) [Neonatal ulcerative colitis in a case]. Zhonghua Er Ke Za Zhi 47: 393-394. BORER F (1960) [Segmental colitis of the newborn. A special form of Crohn's disease]. Helv Paediatr Acta 15: 27-70. Kopecký J, Karásek J, Navrátil M (1978) [Crohn's disease as the cause of intestinal obstruction in newborn infants]. Rozhl Chir 57: 656-659. Baldi A, Di Marino MP, Vicidomini G, Baldi F (1998) Crohn's disease in infancy: a case report. Int Surg 83: 154-156. Zhu ML, Lin ZL, Wu BW (2010) [Neonatal Crohn's disease in a case]. Zhonghua Er Ke Za Zhi 48: 474-475. Kappelman MD, Grand RJ (2008) Does inflammatory bowel disease develop in infants? Inflamm Bowel Dis 14 Suppl 2: S6-8. Rukunuzzaman M, Karim AS (2011) Ulcerative colitis in infancy. Saudi J Gastroenterol 17: 414-417. Holland N, Dong J, Garnett E, Shaikh N, Huen K, et al. (2008) Reduced intracellular T-helper 1 interferon gamma in blood of newly diagnosed children with Crohn’s disease and age-related changes in Th1/Th2 cytokine profiles. Pediatr Res 63: 257-262. Liesenfeld O, Kosek JC, Suzuki Y (1997) Gamma interferon induces Fas-dependent apoptosis of Peyer's patch T cells in mice following peroral infection with Toxoplasma gondii. Infect Immun 65: 4682-4689. Suzuki Y, Orellana MA, Schreiber RD, Remington JS (1988) Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 240: 516-518. Butcher BA, Greene RI, Henry SC, Annecharico KL, Weinberg JB, et al. (2005) p47 GTPases regulate Toxoplasma gondii survival in activated macrophages. Infect Immun 73: 3278-3286. Thouvenin M, Candolfi E, Villard O, Klein JP, Kien T (1997) Immune response in a murine model of congenital toxoplasmosis: increased susceptibility of pregnant mice and transplacental passage of Toxoplasma gondii are type 2-dependent. Parassitologia 39: 279-283. Shiono Y, Mun HS, He N, Nakazaki Y, Fang H, et al. (2007) Maternal-fetal transmission of Toxoplasma gondii in interferon-gamma deficient pregnant mice. Parasitol Int 56: 141-148. Gavrilescu LC, Denkers EY (2001) IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. J Immunol 167: 902-909. Nguyen TD, Bigaignon G, Markine-Goriaynoff D, Heremans H, Nguyen TN, et al. (2003) Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and gamma-interferon. J Med Microbiol 52: 869-876. Thomas MR, Williamson R, Craft I, Yazdani N, Rodeck CH (1994) Y chromosome sequence DNA amplified from peripheral blood of women in early pregnancy. Lancet 343: 413-414. Khosrotehrani K, Bianchi DW (2003) Fetal cell microchimerism: helpful or harmful to the parous woman? Curr Opin Obstet Gynecol 15: 195-199. Schröder J, Tiilikainen A, De la Chapelle A (1974) Fetal leukocytes in the maternal circulation after delivery. I. Cytological aspects. Transplantation 17: 346-354. Khosrotehrani K, Johnson KL, Cha DH, Salomon RN, Bianchi DW (2004) Transfer of fetal cells with multilineage potential to maternal tissue. JAMA 292: 75-80. Khosrotehrani K, Bianchi DW (2005) Multi-lineage potential of fetal cells in maternal tissue: a legacy in reverse. J Cell Sci 118: 1559-1563. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA (1996) Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci U S A 93: 705-708. O'Donoghue K, Chan J, de la Fuente J, Kennea N, Sandison A, et al. (2004) Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy. Lancet 364: 179-182. Owen RD (1945) IMMUNOGENETIC CONSEQUENCES OF VASCULAR ANASTOMOSES BETWEEN BOVINE TWINS. Science 102: 400-401. van Dijk BA, Boomsma DI, de Man AJ (1996) Blood group chimerism in human multiple births is not rare. Am J Med Genet 61: 264-268. Klonisch T, Drouin R (2009) Fetal-maternal exchange of multipotent stem/progenitor cells: microchimerism in diagnosis and disease. Trends Mol Med 15: 510-518. Zhou L, Yoshimura Y, Huang Y, Suzuki R, Yokoyama M, et al. (2000) Two independent pathways of maternal cell transmission to offspring: through placenta during pregnancy and by breast-feeding after birth. Immunology 101: 570-580. Molitor ML, Haynes LD, Jankowska-Gan E, Mulder A, Burlingham WJ (2004) HLA class I noninherited maternal antigens in cord blood and breast milk. Hum Immunol 65: 231-239. Parant O, Dubernard G, Challier JC, Oster M, Uzan S, et al. (2009) CD34+ cells in maternal placental blood are mainly fetal in origin and express endothelial markers. Lab Invest 89: 915-923. Artlett CM (2005) Pathophysiology of fetal microchimeric cells. Clin Chim Acta 360: 1-8. Srivatsa B, Srivatsa S, Johnson KL, Bianchi DW (2003) Maternal cell microchimerism in newborn tissues. J Pediatr 142: 31-35. Prandota J (2011) Metabolic, immune, epigenetic, andocrine and phenotypic abnormalities found in individuals with autism spectrum disorders, Down syndrome and Alzheimer disease may be caused by congenital and/or acquired chronic cerebral toxoplasmosis. Res Autism Spectr Disord 5: 14-59. Leveque L, Khosrotehrani K (2011) Can maternal microchimeric cells influence the fetal response toward self antigens. Chimerism 2: 71-77. Fanning PA, Jonsson JR, Clouston AD, Edwards-Smith C, Balderson GA, et al. (2000) Detection of male DNA in the liver of female patients with primary biliary cirrhosis. J Hepatol 33: 690-695. Zhang B, Angelidou A, Alysandratos KD, Vasiadi M, Francis K, et al. (2010) Mitochondrial DNA and anti-mitochondrial antibodies in serum of autistic children. J Neuroinflammation 7: 80. Bidgoli S, Koch P, Caspers L (2011) [Toxoplasmic chorioretinitis: positive PCR on vitreous with negative serology for Toxoplasma gondii]. J Fr Ophtalmol 34: 384. Nahmias AJ, Nahmias SB, Danielsson D (2006) The possible role of transplacentally-acquired antibodies to infectious agents, with molecular mimicry to nervous system sialic acid epitopes, as causes of neuromental disorders: Prevention and vaccine implications. Clin Dev Immunol 13: 167-183. Van de Water J, Cooper A, Surh CD, Coppel R, Danner D, et al. (1989) Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 320: 1377-1380. Artlett CM, Smith JB, Jimenez SA (1998) Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis. N Engl J Med 338: 1186-1191. Artlett CM, Cox LA, Ramos RC, Dennis TN, Fortunato RA, et al. (2002) Increased microchimeric CD4+ T lymphocytes in peripheral blood from women with systemic sclerosis. Clin Immunol 103: 303-308. Reed AM, Picornell YJ, Harwood A, Kredich DW (2000) Chimerism in children with juvenile dermatomyositis. Lancet 356: 2156-2157. Artlett CM, Ramos R, Jiminez SA, Patterson K, Miller FW, et al. (2000) Chimeric cells of maternal origin in juvenile idiopathic inflammatory myopathies. Childhood Myositis Heterogeneity Collaborative Group. Lancet 356: 2155-2156. Abbud Filho M, Pavarino-Bertelli EC, Alvarenga MP, Fernandes IM, Toledo RA, et al. (2002) Systemic lupus erythematosus and microchimerism in autoimmunity. Transplant Proc 34: 2951-2952. Endo T, Pelster B, Piekarski G (1981) Infection of murine peritoneal macrophages with Toxoplasma gondii exposed to ultraviolet light. Z Parasitenkd 65: 121-129. Klintschar M, Schwaiger P, Mannweiler S, Regauer S, Kleiber M (2001) Evidence of fetal microchimerism in Hashimoto's thyroiditis. J Clin Endocrinol Metab 86: 2494-2498. Ando T, Imaizumi M, Graves PN, Unger P, Davies TF (2002) Intrathyroidal fetal microchimerism in Graves' disease. J Clin Endocrinol Metab 87: 3315-3320. Vabres P, Malinge MC, Larrègue M, Bonneau D (2002) Microchimerism from a dizygotic twin in juvenile ulcerative lichen planus. Lancet 359: 1861-1862. Aractingi S, Berkane N, Bertheau P, Le Goué C, Dausset J, et al. (1998) Fetal DNA in skin of polymorphic eruptions of pregnancy. Lancet 352: 1898-1901. Nelson JL, Gillespie KM, Lambert NC, Stevens AM, Loubiere LS, et al. (2007) Maternal microchimerism in peripheral blood in type 1 diabetes and pancreatic islet beta cell microchimerism. Proc Natl Acad Sci U S A 104: 1637-1642. Wrenshall LE, Stevens ET, Smith DR, Miller JD (2007) Maternal microchimerism leads to the presence of interleukin-2 in interleukin-2 knock out mice: implications for the role of interleukin-2 in thymic function. Cell Immunol 245: 80-90. Hayashida M, Nishimoto Y, Matsuura T, Takahashi Y, Kohashi K, et al. (2007) The evidence of maternal microchimerism in biliary atresia using fluorescent in situ hybridization. J Pediatr Surg 42: 2097-2101. Muraji T, Hosaka N, Irie N, Yoshida M, Imai Y, et al. (2008) Maternal microchimerism in underlying pathogenesis of biliary atresia: quantification and phenotypes of maternal cells in the liver. Pediatrics 121: 517-521. Tanaka A, Lindor K, Gish R, Batts K, Shiratori Y, et al. (1999) Fetal microchimerism alone does not contribute to the induction of primary biliary cirrhosis. Hepatology 30: 833-838. Schöniger-Hekele M, Müller C, Ackermann J, Drach J, Wrba F, et al. (2002) Lack of evidence for involvement of fetal microchimerism in pathogenesis of primary biliary cirrhosis. Dig Dis Sci 47: 1909-1914. Glauben R, Batra A, Fedke I, Zeitz M, Lehr HA, et al. (2006) Histone hyperacetylation is associated with amelioration of experimental colitis in mice. J Immunol 176: 5015-5022. Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, et al. (1997) A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med 337: 1029-1035. Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W (2002) Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17: 629-638. Strobl JS, Cassell M, Mitchell SM, Reilly CM, Lindsay DS (2007) Scriptaid and suberoylanilide hydroxamic acid are histone deacetylase inhibitors with potent anti-Toxoplasma gondii activity in vitro. J Parasitol 93: 694-700. Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403: 41-45. Takai N, Desmond JC, Kumagai T, Gui D, Said JW, et al. (2004) Histone deacetylase inhibitors have a profound antigrowth activity in endometrial cancer cells. Clin Cancer Res 10: 1141-1149. Spencer VA, Davie JR (1999) Role of covalent modifications of histones in regulating gene expression. Gene 240: 1-12. MacDonald JL, Roskams AJ (2009) Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation. Prog Neurobiol 88: 170-183. Mishra N, Reilly CM, Brown DR, Ruiz P, Gilkeson GS (2003) Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest 111: 539-552. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, et al. (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 276: 36734-36741. Leoni F, Zaliani A, Bertolini G, Porro G, Pagani P, et al. (2002) The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines. Proc Natl Acad Sci USA 99: 2995-3000. Darkin-Rattray SJ, Gurnett AM, Myers RW, Dulski PM, Crumley TM, et al. (1996) Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase. Proc Natl Acad Sci USA 93: 13143-13147. Prandota J (2009) The importance of toxoplasma gondii infection in diseases presenting with headaches. Headaches and aseptic meningitis may be manifestations of the Jarisch-Herxheimer reaction. Int J Neurosci 119: 2144-2182. Kusbeci OY, Miman O, Yaman M, Aktepe OC, Yazar S (2011) Could Toxoplasma gondii have any role in Alzheimer disease? Alzheimer Dis Assoc Disord 25: 1-3. Ren M, Leng Y, Jeong M, Leeds PR, Chuang DM (2004) Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction. J Neurochem 89: 1358-1367. Duffy M, O'Mahony L, Coffey JC, Collins JK, Shanahan F, et al. (2002) Sulfate-reducing bacteria colonize pouches formed for ulcerative colitis but not for familial adenomatous polyposis. Dis Colon Rectum 45: 384-388. Ohge H, Furne JK, Springfield J, Rothenberger DA, Madoff RD, et al. (2005) Association between fecal hydrogen sulfide production and pouchitis. Dis Colon Rectum 48: 469-475. Mutsaers SE, Papadimitriou JM (1988) Surface charge of macrophages and their interaction with charged particles. J Leukoc Biol 44: 17-26. Cintra WM, Silva-Filho FC, De Souza W (1986) The surface charge of Toxoplasma gondii: a cytochemical and electrophoretic study. J Submicrosc Cytol 18: 773-781. Guimarães EV, Acquarone M, de Carvalho L, Barbosa HS (2007) Anionic sites on Toxoplasma gondii tissue cyst wall: expression, uptake and characterization. Micron 38: 651-658. Carruthers VB, Håkansson S, Giddings OK, Sibley LD (2000) Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment. Infect Immun 68: 4005-4011. Ortega-Barria E, Boothroyd JC (1999) A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection. J Biol Chem 274: 1267-1276. Monteiro VG, Soares CP, de Souza W (1998) Host cell surface sialic acid residues are involved on the process of penetration of Toxoplasma gondii into mammalian cells. FEMS Microbiol Lett 164: 323-327. Bishop JR, Esko JD (2005) The elusive role of heparan sulfate in Toxoplasma gondii infection. Mol Biochem Parasitol 139: 267-269. Esko JD, Lindahl U (2001) Molecular diversity of heparan sulfate. J Clin Invest 108: 169-173. Jacquet A, Coulon L, De Nève J, Daminet V, Haumont M, et al. (2001) The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans. Mol Biochem Parasitol 116: 35-44. Coppi A, Tewari R, Bishop JR, Bennett BL, Lawrence R, et al. (2007) Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells. Cell Host Microbe 2: 316-327. Compton T, Nowlin DM, Cooper NR (1993) Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193: 834-841. Alvarez-Dominguez C, Vazquez-Boland JA, Carrasco-Marin E, Lopez-Mato P, Leyva-Cobian F (1997) Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface ActA is involved in heparan sulfate receptor rcognition. Infect Immun 65: 78-88. Spear PG, Shieh MT, Herold BC, WuDunn D, Koshy TI (1992) Heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus. Adv Exp Med Biol 313: 341-353. Dvorak AM, Onderdonk AB, McLeod RS, Monahan-Earley RA, Cullen J, et al. (1993) Axonal necrosis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn's disease. Ann Surg 217: 260-271. Simpson J, Sundler F, Humes DJ, Jenkins D, Scholefield JH, et al. (2009) Post inflammatory damage to the enteric nervous system in diverticular disease and its relationship to symptoms. Neurogastroenterol Motil 21: 847-847e58. Kieslich M, Errázuriz G, Posselt HG, Moeller-Hartmann W, Zanella F, et al. (2001) Brain white-matter lesions in celiac disease: a prospective study of 75 diet-treated patients. Pediatrics 108: E21. Roche Herrero MC, Arcas Martínez J, Martínez-Bermejo A, López Martín V, Polanco I, et al. (2001) [The prevalence of headache in a population of patients with coeliac disease]. Rev Neurol 32: 301-309. Arroyo HA, De Rosa S, Ruggieri V, de Dávila MT, Fejerman N; Argentinean Epilepsy and Celiac Disease Group (2002) Epilepsy, occipital calcifications, and oligosymptomatic celiac disease in childhood. J Child Neurol 17: 800-806. Gabrielli M, Cremonini F, Fiore G, Addolorato G, Padalino C, et al. (2003) Association between migraine and Celiac disease: results from a preliminary case-control and therapeutic study. Am J Gastroenterol 98: 625-629. Zelnik N, Pacht A, Obeid R, Lerner A (2004) Range of neurologic disorders in patients with celiac disease. Pediatrics 113: 1672-1676. Pfaender M, D'Souza WJ, Trost N, Litewka L, Paine M, et al. (2004) Visual disturbances representing occipital lobe epilepsy in patients with cerebral calcifications and coeliac disease: a case series. J Neurol Neurosurg Psychiatry 75: 1623-1625. Bushara KO (2005) Neurologic presentation of celiac disease. Gastroenterology 128: S92-97. Hu WT, Murray JA, Greenaway MC, Parisi JE, Josephs KA (2006) Cognitive impairment and celiac disease. Arch Neurol 63: 1440-1446. Hansson T, Dannaeus A, Klareskog L (1999) Cytokine-producing cells in peripheral blood of children with coeliac disease secrete cytokines with a type 1 profile. Clin Exp Immunol 116: 246-250. Tucková L, Flegelová Z, Tlaskalová-Hogenová H, Zídek Z (2000) Activation of macrophages by food antigens: enhancing effect of gluten on nitric oxide and cytokine production. J Leukoc Biol 67: 312-318. Lahat N, Shapiro S, Karban A, Gerstein R, Kinarty A, et al. (1999) Cytokine profile in coeliac disease. Scand J Immunol 49: 441-446. Ertekin V, Selimoglu MA, Turkan Y, Akcay F (2005) Serum nitric oxide levels in children with celiac disease. J Clin Gastroenterol 39: 782-785. Gobbi G (2005) Coeliac disease, epilepsy and cerebral calcifications. Brain Dev 27: 189-200. Wills AJ (2000) The neurology and neuropathology of coeliac disease. Neuropathol Appl Neurobiol 26: 493-496. Fois A, Vascotto M, Di Bartolo RM, Di Marco V (1994) Celiac disease and epilepsy in pediatric patients. Childs Nerv Syst 10: 450-454. Ambrosetto G, Antonini L, Tassinari CA (1992) Occipital lobe seizures related to clinically asymptomatic celiac disease in adulthood. Epilepsia 33: 476-481. Díaz RM, González-Rabelino G, Delfino A (2005) [Epilepsy, cerebral calcifications and coeliac disease. The importance of an early diagnosis]. Rev Neurol 40: 417-420. Vuppugalla R, Mehvar R (2004) Hepatic disposition and effects of nitric oxide donors: rapid and concentration dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers. J Pharmacol Exp Ther 310: 718-727. Rostami Nejad M, Rostami K, Cheraghipour K, Nazemalhosseini Mojarad E, Volta U, et al. (2011) Celiac disease increases the risk of Toxoplasma gondii infection in a large cohort of pregnant women. Am J Gastroenterol 106: 548-549. Verzegnassi F, Bua J, De Angelis P, Dall'oglio L, Di Leo G, et al. (2007) Eosinophilic oesophagitis and coeliac disease: is it just a casual association? Gut 56: 1029-1030. Ooi CY, Day AS, Jackson R, Bohane TD, Tobias V, et al. (2008) Eosinophilic esophagitis in children with celiac disease. J Gastroenterol Hepatol 23: 1144-1148. Heine RG (2008) Eosinophilic esophagitis in children with celiac disease: new diagnostic and therapeutic dilemmas. J Gastroenterol Hepatol 23: 993-994. Józefczuk J, Woźniewicz BM (2011) Diagnosis and therapy of microscopic colitis with presence of foamy macrophages in children. ISRN Gastroenterol 2011: 756292. Jozefczuk J, Wozniewicz B, Romanczuk W (2001) Clinicopathology of foamy cell colitis (FCC), the new form of microscopic colitis in children. Ann Diagn Paediatr Pathol 5: 71-74. Józefczuk J, Wozniewicz BM (2008) Clear cell colitis: a form of microscopic colitis in children. World J Gastroenterol 14: 231-235. Yantiss RK, Odze RD (2006) Diagnostic difficulties in inflammatory bowel disease pathology. Histopathology 48: 116-132. Russell DG, Cardona PJ, Kim MJ, Allain S, Altare F (2009) Foamy macrophages and the progression of the human tuberculosis granuloma. Nat Immunol 10: 943-948. Pettersen EK (1979) Destruction of Toxoplasma gondii by HC1 solution. Acta Pathol Microbiol Scand B 87: 217-220. Sharma SP, Dubey JP (1981) Quantitative survival of Toxoplasma gondii tachyzoites and bradyzoites in pepsin and in trypsin solutions. Am J Vet Res 42: 128-130. Popiel I, Gold MC, Booth KS (1996) Quantification of Toxoplasma gondii bradyzoites. J Parasitol 82: 330-332. Dubey JP (1998) Re-examination of resistance of Toxoplasma gondii tachyzoites and bradyzoites to pepsin and trypsin digestion. Parasitology 116 : 43-50. Reed MD (1992) Principles of drug therapy. Behrman RE, Kliegman RM, Nelson WE, Vaughan VC (eds), Nelson Textbook of Pediatrics, (14thedn), WB Saunders Co, Philadelphia 252-258. Nishikawa Y, Quittnat F, Stedman TT, Voelker DR, Choi JY, et al. (2005) Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma. Cell Microbiol 7: 849-867. D'Avila H, Maya-Monteiro CM, Bozza PT (2008) Lipid bodies in innate immune response to bacterial and parasite infections. Int Immunopharmacol 8: 1308-1315. Silva AR, Pacheco P, Vieira-de-Abreu A, Maya-Monteiro CM, D’Alegria B, et al. (2009) Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: A MCP-1/CCL2 regulated phenomenon. Biochem Biophys Acta 179: 1066-1075. D'Avila H, Melo RC, Parreira GG, Werneck-Barroso E, Castro-Faria-Neto HC, et al. (2006) Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo. J Immunol 176: 3087-3097. Goldberg HI, Gore RM, Margulis AR, Moss AA, Baker EL (1983) Computed tomography in the evaluation of Crohn disease. AJR Am J Roentgenol 140: 277-282. Nagamata H, Inadama E, Arihiro S, Matsuoka M, Torii A, et al. (2002) [The usefulness of MDCT in Crohn's disease]. Nihon Shokakibyo Gakkai Zasshi 99: 1317-1325. Lucey BC, Stuhlfaut JW, Soto JA (2005) Mesenteric lymph nodes seen at imaging: causes and significance. Radiographics 25: 351-365. Sivit CJ, Newman KD, Chandra RS (1993) Visualization of enlarged mesenteric lymph nodes at US examination. Clinical significance. Pediatr Radiol 23: 471-475. Vayner N, Coret A, Polliack G, Weiss B, Hertz M (2003) Mesenteric lymphadenopathy in children examined by US for chronic and/or recurrent abdominal pain. Pediatr Radiol 33: 864-867. Karmazyn B, Werner EA, Rejaie B, Applegate KE (2005) Mesenteric lymph nodes in children: what is normal? Pediatr Radiol 35: 774-777. Rathaus V, Shapiro M, Grunebaum M, Zissin R (2005) Enlarged mesenteric lymph nodes in asymptomatic children: the value of the finding in various imaging modalities. Br J Radiol 78: 30-33. Simanovsky N, Hiller N (2007) Importance of sonographic detection of enlarged abdominal lymph nodes in children. J Ultrasound Med 26: 581-584. WANG WG, TIAN H, YAN JY, LI T, ZHANG TD, et al. (2011) [Enlarged mesenteric lymph nodes in children: a clinical analysis with ultrasonography and the implications]. Nan Fang Yi Ke Da Xue Xue Bao 31: 522-524. Watanabe M, Ishii E, Hirowatari Y, Hayashida Y, Koga T, et al. (1997) Evaluation of abdominal lymphadenopathy in children by ultrasonography. Pediatr Radiol 27: 860-864. Birnbaum BA, Jeffrey RB Jr (1998) CT and sonographic evaluation of acute right lower quadrant abdominal pain. AJR Am J Roentgenol 170: 361-371. Zenner L, Darcy F, Capron A, Cesbron-Delauw MF (1998) Toxoplasma gondii: kinetics of the dissemination in the host tissues during the acute phase of infection of mice and rats. Exp Parasitol 90: 86-94. Kodjikian L, Hoigne I, Adam O, Jacquier P, Aebi-Ochsner C, et al. (2004) Vertical transmission of toxoplasmosis from a chronically infected immunocompetent woman. Pediatr Infect Dis J 23: 272-274. Bonametti AM, Passos JN, Koga da Silva EM, Macedo ZS (1997) Probable transmission of acute toxoplasmosis through breast feeding. J Trop Pediatr 43: 116. Howie PW, Forsyth JS, Ogston SA, Clark A, Florey CD (1990) Protective effect of breast feeding against infection. BMJ 300: 11-16. Milosavljević N, Virijević V (1997) [Methods of feeding and illness in infants in the first six months of life]. Srp Arh Celok Lek 125: 325-328. Pesonen M, Kallio MJ, Ranki A, Siimes MA (2006) Prolonged exclusive breastfeeding is associated with increased atopic dermatitis: a prospective follow-up study of unselected healthy newborns from birth to age 20 years. Clin Exp Allergy 36: 1011-1018. Saarinen UM, Kajosaari M (1995) Breastfeeding as prophylaxis against atopic disease: prospective follow-up study until 17 years old. Lancet 346: 1065-1069. Goldman AS, Thorpe LW, Goldblum RM, Hanson LA (1986) Anti-inflammatory properties of human milk. Acta Paediatr Scand 75: 689-695. Hooton JW, Pabst HF, Spady DW, Paetkau V (1991) Human colostrum contains an activity that inhibits the production of IL-2. Clin Exp Immunol 86: 520-524. Mandalapu P, Pabst HF, Paetkau V (1995) A novel immunosuppressive factor in human colostrum. Cell Immunol 162: 178-184. Goldman AS, Garza C, Nichols BL, Goldblum RM (1982) Immunologic factors in human milk during the first year of lactation. J Pediatr 100: 563-567. Legrand D, Elass E, Pierce A, Mazurier J (2004) Lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties. Biometals 17: 225-229. Houghton MR, Gracey M, Burke V, Bottrell C, Spargo RM (1985) Breast milk lactoferrin levels in relation to maternal nutritional status. J Pediatr Gastroenterol Nutr 4: 230-233. Masson PL, Heremans JF, Schonne E (1969) Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J Exp Med 130: 643-658. Maaks S, Yan HZ, Wood WG (1989) Development and evaluation of luminescence based sandwich assay for plasma lactoferrin as a marker for sepsis and bacterial infections in pediatric medicine. J Biolumin Chemilumin 3: 221-226. Pacora P, Maymon E, Gervasi MT, Gomez R, Edwin SS, et al. (2000) Lactoferrin in intrauterine infection, human parturition, and rupture of fetal membranes. Am J Obstet Gynecol 183: 904-910. Lönnerdal B, Iyer S (1995) Lactoferrin: molecular structure and biological function. Annu Rev Nutr 15: 93-110. Ziere GJ, Van Dijk MCM, Bijsterbosch MK, Van Berkel TJC (1992) Lactoferrin uptake by the rat liver: characterization of the recognition site and effect of selective modification of arginine residues. J Biol Chem 267: 11229-11235. Bennatt DJ, Ling YY, McAbee DD (1997) Isolated rat hepatocytes bind lactoferrins by the RHL-1 subunit of the asialoglycoprotein receptor in a galactose-independent manner. Biochemistry 36: 8367-8376. Mann DM, Romm E, Migliorini M (1994) Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin. J Biol Chem 269: 23661-23667. van der Strate BW, Beljaars L, Molema G, Harmsen MC, Meijer DK (2001) Antiviral activities of lactoferrin. Antiviral Res 52: 225-239. Baveye S, Elass E, Mazurier J, Legrand D (2000) Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils. FEBS Lett 469: 5-8. Pabst HF, Spady DW, Pilarski LM, Carson MM, Beeler JA, et al. (1997) Differential modulation of the immune response by breast- or formula-feeding of infants. Acta Paediatr 86: 1291-1297. Legrand D, Elass E, Carpentier M, Mazurier J (2005) Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 62: 2549-2559. Hirotani Y, Ikeda K, Kato R, Myotoku M, Umeda T, et al. (2008) Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells. Yakugaku Zasshi 128: 1363-1368. Legrand D (2012) Lactoferrin, a key molecule in immune and inflammatory processes. Biochem Cell Biol 90: 252-268. Baveye S, Elass E, Fernig DG, Blanquart C, Mazurier J, et al. (2000) Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex. Infect Immun 68: 6519-6525. Machnicki M, Zimecki M, Zagulski T (1993) Lactoferrin regulates the release of tumour necrosis factor alpha and interleukin 6 in vivo. Int J Exp Pathol 74: 433-439. Kruzel ML, Harari Y, Mailman D, Actor JK, Zimecki M (2002) Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol 130: 25-31. Sawatzki G, Rich IN (1989) Lactoferrin stimulates colony stimulating factor production in vitro and in vivo. Blood Cells 15: 371-385. Sorimachi K, Akimoto K, Hattori Y, Ieiri T, Niwa A (1997) Activation of macrophages by lactoferrin: secretion of TNF-alpha, IL-8 and NO. Biochem Mol Biol Int 43: 79-87. Shimizu K, Matsuzawa H, Okada K, Tazume S, Dosako S, et al. (1996) Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity. Arch Virol 141: 1875-1889. Shau H, Kim A, Golub SH (1992) Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin. J Leukoc Biol 51: 343-349. Yamauchi K, Wakabayashi H, Hashimoto S, Teraguchi S, Hayasawa H, et al. (1998) Effects of orally administered bovine lactoferrin on the immune system of healthy volunteers. Adv Exp Med Biol 443: 261-265. Szuster-Ciesielska A, KamiÅ„ska T, Kandefer-SzerszeÅ„ M (1995) Phagocytosis-enhancing effect of lactoferrin on bovine peripheral blood monocytes in vitro and in vivo. Arch Vet Pol 35: 63-71. Togawa J, Nagase H, Tanaka K, Inamori M, Nakajima A, et al. (2002) Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. J Gastroenterol Hepatol 17: 1291-1298. Håversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I (2002) Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 220: 83-95. Na YJ, Han SB, Kang JS, Yoon YD, Park SK, et al. (2004) Lactoferrin works as a new LPS-binding protein in inflammatory activation of macrophages. Int Immunopharmacol 4: 1187-1199. Artym J, Zimecki M, Paprocka M, Kruzel ML (2003) Orally administered lactoferrin restores humoral immune response in immunocompromised mice. Immunol Lett 89: 9-15. Ambruso DR, Johnston RB Jr (1981) Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system. J Clin Invest 67: 352-360. Raghuveer TS, McGuire EM, Martin SM, Wagner BA, Rebouché CJ, et al. (2002) Lactoferrin in the preterm infants' diet attenuates iron-induced oxidation products. Pediatr Res 52: 964-972. Davidsson L, Kastenmayer P, Yuen M, Lönnerdal B, Hurrell RF (1994) Influence of lactoferrin on iron absorption from human milk in infants. Pediatr Res 35: 117-124. Kawakami H, Lönnerdal B (1991) Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes. Am J Physiol 261: G841-846. Fillebeen C, Ruchoux MM, Mitchell V, Vincent S, Benaïssa M, et al. (2001) Lactoferrin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment. Brain Res Mol Brain Res 96: 103-113. Fillebeen C, Dehouck B, Benaïssa M, Dhennin-Duthille I, Cecchelli R, et al. (1999) Tumor necrosis factor-alpha increases lactoferrin transcytosis through the blood-brain barrier. J Neurochem 73: 2491-2500. Hammer J, Haaheim H, Gutteberg TJ (2000) Bovine lactoferrin is more efficient than bovine lactoferricin in inhibiting HSV-I/-II replication in vitro. Shimazaki K (ed), Lactoferrin: Structure, Functions and Applications. Elsevier Science, Amsterdam, 239-243. Nanau RM, Neuman MG (2012) Metabolome and inflammasome in inflammatory bowel disease. Transl Res 160: 1-28. Pfefferkorn MD, Boone JH, Nguyen JT, Juliar BE, Davis MA, et al. (2010) Utility of fecal lactoferrin in identifying Crohn disease activity in children. J Pediatr Gastroenterol Nutr 51: 425-428. Dai J, Liu WZ, Zhao YP, Hu YB, Ge ZZ (2007) Relationship between fecal lactoferrin and inflammatory bowel disease. Scand J Gastroenterol 42: 1440-1444. Sidhu R, Wilson P, Wright A, Yau CW, D'Cruz FA, et al. (2010) Faecal lactoferrin--a novel test to differentiate between the irritable and inflamed bowel? Aliment Pharmacol Ther 31: 1365-1370. Bout D, Moretto M, Dimier-Poisson I, Gatel DB (1999) Interaction between Toxoplasma gondii and enterocyte. Immunobiology 201: 225-228. Chardès T, Buzoni-Gatel D, Lepage A, Bernard F, Bout D (1994) Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes. J Immunol 153: 4596-4603. Dimier IH, Bout DT (1998) Interferon-gamma-activated primary enterocytes inhibit Toxoplasma gondii replication: a role for intracellular iron. Immunology 94: 488-495. Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat 141: 461-479. Brandtzaeg P (1974) Mucosal and glandular distribution of immunoglobulin components: differential localization of free and bound SC in secretory epithelial cells. J Immunol 112: 1553-1559. Qian ZM, Tang PL (1995) Mechanisms of iron uptake by mammalian cells. Biochim Biophys Acta 1269: 205-214. Newburg DS, Ruiz-Palacios GM, Morrow AL (2005) Human milk glycans protect infants against enteric pathogens. Ann Rev Nutr 25: 37-58. Newburg DS (1997) Do the binding properties of oligosaccharides in milk protect human infants from gastrointestinal bacteria? J Nutr 127: 980S-984S. Sisk PM, Lovelady CA, Dillard RG, Gruber KJ, O’Shea TM (2007) Early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants. J Perinatol 27: 428-433. Sherman MP, Bennett SH, Hwang FF, Yu C (2004) Neonatal small bowel epithelia: enhancing anti-bacterial defense with lactoferrin and Lactobacillus GG. Biometals 17: 285-289. Sherman MP, Petrak K (2005) Lactoferrin-enhanced anoikis: a defense against neonatal necrotizing enterocolitis. Med Hypotheses 65: 478-482. Isamida T, Tanaka T, Omata Y, Yamauchi K, Shimazaki K, et al. (1998) Protective effect of lactoferrin against Toxoplasma gondii infection in mice. J Vet Med Sci 60: 241-244. Omata Y, Satake M, Maeda R, Saito A, Shimazaki K, et al. (2001) Reduction of the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoites by treatment with bovine lactoferricin. J Vet Med Sci 63: 187-190. Tanaka T, Omata Y, Isamida T, Saito A, Shimazaki K, et al. (1998) Growth inhibitory effect of bovine lactoferrin to Toxoplasma gondii tachyzoites in murine macrophages: tyrosine phosphorylation in murine macrophages induced by bovine lactoferrin. J Vet Med Sci 60: 369-371. Dzitko K, Dziadek B, Dziadek J, DÅ‚ugoÅ„ska H (2007) Toxoplasma gondii: inhibition of the intracellular growth by human lactoferrin. Pol J Microbiol 56: 25-32. Tanaka T, Omata Y, Saito A, Shimazaki K, Igarashi I, et al. (1996) Growth inhibitory effects of bovine lactoferrin to Toxoplasma gondii parasites in murine somatic cells. J Vet Med Sci 58: 61-65. Ryu E (1982) Prophylactic effect of tea on pathogenic microorganism infections to humans and animals. (II). Protozoacidal effect on Toxoplasma gondii in vitro and mice. Int J Zoonoses 9: 126-131.