Virology & Mycology Infectious Diseases
 
   Publications A-Z
A
»Accounting & Marketing
» Addiction Research & Therapy
» Advanced Chemical Engineering
»Advances in Automobile Engineering
»Advances in Crop Science and Technology
»Advances in Dairy Research
»Advancements in Genetic Engineering
»Advances in Pharmacoepidemiology & Drug Safety
»Advances in Robotics & Automation
»Advanced Techniques in Biology & Medicine
»Advance Research in Meteorological Sciences
»Aeronautics & Aerospace Engineering
»Aging Science
»Agrotechnology
»AIDS & Clinical Research
»Alcoholism and Drug Dependence
»Allergy & Therapy
»Alternative & Integrative Medicine
»Air & Water borne Diseases
»Alzheimers Disease & Parkinsonism
»Analytical & Bioanalytical Techniques
»Anaplastology
»Anatomy & Physiology
»Ancient Diseases & Preventive Remedies
»Andrology-Open Access
»Anesthesia & Clinical Research
»Angiology: Open Access
»Antivirals & Antiretrovirals
»Anthropology
»Applied & Computational Mathematics
»Applied Mechanical Engineering
»Aquaculture Research & Development
» Arabian Journal Business and Management Review
»Architectural Engineering Technology
»Arthritis
»Arts and Social Sciences Journal
» Astrobiology & Outreach
»Astrophysics & Aerospace technology
»Autacoids
»Automatic Control of Physiological State and Function
»Autism-Open Access
  
B
»Bacteriology & Parasitology
»Bioanalysis & Biomedicine
»Bioceramics Development and Applications
»Biochemistry and Analytical Biochemistry
»Biochemistry & Pharmacology: Open Access
»Biochips & Tissue Chips
»Biodiversity & Endangered Species
»Bioenergetics: Open Access
»Bioengineering & Biomedical Science
»Bioequivalence & Bioavailability
»Biosafety & Health Education
»Biofertilizers & Biopesticides
»Biometrics & Biostatistics
»Biomimetics Biomaterials and Tissue Engineering
»Biomolecular Research & Therapeutics
» Biomusical Engineering
»Biochemistry & Physiology: Open Access
»Biological Systems- Open Access
»Biology and Medicine
»Bioprocessing & Biotechniques
»Bioremediation & Biodegradation
»Biosafety
»Biosensors & Bioelectronics
» Biosensors Journal
»Biotechnology & Biomaterials
»Bioterrorism & Biodefense
»Blood Disorders & Transfusion
»Blood & Lymph
»Bone Marrow Research
»Brain Disorders & Therapy
»Business and Economics Journal
»Business and Financial Affairs
  
C
»Cancer Science & Therapy
»Climatology & Weather forecasting
»Carcinogenesis & Mutagenesis
»Cardiovascular Diseases & Diagnosis
»Cardiovascular Pharmacology: Open Access
»Cell & Developmental Biology
»Cell Science & Therapy
»Chemical Engineering & Process Technology
»Chemical Sciences Journal
»Chemotherapy: Open Access
»Child and Adolescent Behavior
»Chromatography & Separation Techniques
»Civil & Environmental Engineering
»Civil & Legal Sciences
»Clinical & Cellular Immunology
»Clinical Case Reports
»Clinical & Experimental Cardiology
»Clinical & Experimental Dermatology Research
»Clinical & Experimental Ophthalmology
»Clinical & Experimental Pathology
»Clinical & Experimental Pharmacology
»Clinical Microbiology: Open Access
»Clinical Pharmacology & Biopharmaceutics
»Clinical Research & Bioethics
»Clinical Research on Foot & Ankle
»Clinical Toxicology
»Clinical Trials
»Clinics in Mother and Child Health
»Cloning & Transgenesis
»Community Medicine & Health Education
»Communication Disorders, Deaf Studies & Hearing Aids
»Computer Science & Systems Biology
» Current Synthetic & Systems Biology
»Cytology & Histology
D
»Data Mining in Genomics & Proteomics
»Defense Management
»Dentistry
»Depression and Anxiety
»Developing Drugs
»Diabetes & Metabolism
»Drug Designing
»Drug Metabolism & Toxicology
  
E
»Earth Science & Climatic Change
»Ecosystem & Ecography
»Endocrinology & Metabolic Syndrome
»Entomology, Ornithology & Herpetology
»Environmental & Analytical Toxicology
»Epidemiology: Open Access
»Emergency Medicine
»Ergonomics
»Electrical & Electronics
»Enzyme Engineering
»Entrepreneurship & Organization Management
  
F
»Family Medicine & Medical Science Research
»Fermentation Technology
»Fertilization: In Vitro - IVF-Worldwide
»Fisheries and Aquaculture Journal
»Fisheries & Livestock Production
»Food Processing & Technology
»Forensic Biomechanics
»Forensic Research
»Forest Research: Open Access
»Fundamentals of Renewable Energy and Applications
»Fungal Genomics & Biology
  
G
»Gastrointestinal & Digestive System
»Genetic Syndromes & Gene Therapy
»Gene Technology
»Generalized Lie Theory and Applications
»Glycobiology
»Glycomics & Lipidomics
»Gynecology& Obstetrics
»General Medicine
»General Practice
»Geography & Natural Disasters
»Geology & Geosciences
»Geophysics & Remote Sensing
»Gerontology & Geriatric Research
»Global Economics
»Global Journal of Technology and Optimization
  
H
»Hair : Therapy & Transplantation
»Hematology & Thromboembolic Diseases
»Health Care : Current Reviews
»Health & Medical Informatics
»Hereditary Genetics
»Homeopathy & Ayurvedic Medicine
»Horticulture
»Hotel & Business Management
»Human Genetics & Embryology
»Hydrology: Current Research
»Hypertension- Open Access
  
I
»Industrial Engineering & Management
»Irrigation and Drainage Systems Engineering
»Infectious Diseases and Therapy
»Information Technology & Software Engineering
»Intellectual Property Rights: Open Access
»Integrative Oncology
»Internal Medicine
»International Journal of Accounting Research
»International Journal of Advanced Innovations, Thoughts & Ideas
»International Journal of Biomedical Data Mining
»International Journal of Economics and Management Science
»International Journal of Sensor Networks and Data Communications
»International Journal of Swarm Intelligence and Evolutionary Computation
»International Journal of waste resources
»International Journal of Genomic Medicine
»International Journal of Physical Medicine & Rehabilitation
»Innovative Energy Policies
»Immunome Research
  
J
»JBR Journal of Interdisciplinary Medicine and Dental Science
»JBR Journal of Clinical Diagnosis and Research
»JBR Journal of Young Scientist
  
L
»Liver
» Leukemia
» Lovotics
  
M
»Marine Science: Research & Development
»Malaria Chemotherapy, Control & Elimination
»Mass Communication & Journalism
»Material Sciences & Engineering
»Medicinal Chemistry
»Medicinal & Aromatic Plants
»Medical Diagnostic Methods
»Medical Microbiology & Diagnosis
»Medical & Surgical Urology
»Membrane Science & Technology
»Metabolic Syndrome
»Metabolomics:Open Access
»Microbial & Biochemical Technology
»Modern Chemistry & Applications
»Molecular Biology
»Molecular Biomarkers & Diagnosis
»Molecular and Genetic Medicine
»Molecular Imaging & Dynamics
»Molecular Pharmaceutics & Organic Process Research
»Mycobacterial Diseases
N
»Natural Products Chemistry & Research
»Nanomedicine & Biotherapeutic Discovery
»Nanomedicine & Nanotechnology
»Neonatal Biology
»Nephrology & Therapeutics
»Neurology & Neurophysiology
»Neuroinfectious Diseases
»Neurological Disorders
»Novel Physiotherapies
»Nuclear Medicine & Radiation Therapy
»Nutrition & Food Sciences
»Nutritional Disorders & Therapy
»Nursing & Care
  
O
»Obesity & Weight Loss Therapy
»Oceanography-Open Access
»Occupational Medicine & Health Affairs
»OMICS Journal of Radiology
»Osteoporosis and Physical Activity
»Oral Health and Dental Management
»Oral Hygiene & Health
»Organic Chemistry: Current Research
»Orthopedic & Muscular System: Current Research
»Otolaryngology
  
P
»Pain & Relief
»Palliative Care & Medicine
»Pancreatic Disorders & Therapy
»Pediatrics & Therapeutics
»Petroleum & Environmental Biotechnology
»Pharmaceutica Analytica Acta
»Pharmaceutical Regulatory Affairs: Open Access
»Pharmacogenomics & Pharmacoproteomics
»Pharmacovigilance
»Physical Chemistry & Biophysics
»Physical Mathematics
»Plant Pathology & Microbiology
»Plant Biochemistry & Physiology
»Pollution Effects & Control
»political Sciences & public affairs
»Poultry, Fisheries & Wildlife Sciences
»Powder Metallurgy & Mining
»Primatology
»Primary Health Care: Open Access
»Probiotics & Health
» Phylogenetics & Evolutionary Biology
»Proteomics & Bioinformatics
»Phylogenetics & Evolutionary Biology
»Psychology & Psychotherapy
»Psychological Abnormalities in Children
»Pulmonary & Respiratory Medicine
  
R
»Reproductive System & Sexual Disorders
»Research and Development
»Review of Public Adminstration and Management
»Rheumatology: Current Research
»Rice Research: Open Access
  
S
» Single Cell Biology
»Sleep Disorders & Therapy
»Socialomics
»Sociology and Criminology-Open Access
»Sports Medicine & Doping Studies
»Spine
»Stem Cell Research & Therapy
»Steroids & Hormonal Science
»Stock & Forex Trading
»Surgery: Current Research
  
T
»Telecommunications System & Management
»Textile Science & Engineering
»Theoretical and Computational Chemistry:Open Access
»Thermodynamics & Catalysis
»Thyroid Disorders & Therapy
»Tissue Science & Engineering
»Translational Medicine
»Transplantation Technologies & Research
»Transcriptomics: Open Access
»Trauma & Treatment
»Tropical Diseases
»Tropical Medicine and Surgery
»Tourism & Hospitality
  
V
»Vaccines & Vaccination
»Vascular Medicine & surgery
»Veterinary Science & Technology
»Virology & Mycology
»Vitamins & Minerals
»Vortex Science and Technology
  
W
»Women's Health Care
  
Y
»Yoga & Physical Therapy
 
   Browse by Subjects
Clinical
» AIDS & Clinical Research
» Anesthesia & Clinical Research
» Angiology
» Cancer Science & Therapy
» Carcinogenesis & Mutagenesis
» Cell Science & Therapy
» Chemotherapy
» Child & Adolescent Behavior
» Clinical & Cellular Immunology
» Clinical Case Reports
» Clinical & Experimental Cardiology
» Clinical & Experimental Dermatology Research
» Clinical & Experimental Ophthalmology
» Clinical & Experimental Pharmacology
» Clinical & Experimental Pathology
» Clinical Microbiology: Open Access
» Clinical Research & Bioethics
» Clinical Research on Foot & Ankle
» Clinical Toxicology
» Clinical Trials
»Clinics in Mother and Child Health
» Communication Disorders, Deaf Studies & Hearing Aids
» Cytology & Histology
» Forensic Research
»Forensic Biomechanics
» Integrative Oncology
»Immunome Research
»JBR Journal of Clinical Diagnosis and Research
» Neurology & Neurophysiology
» Pulmonary & Respiratory Medicine
» Stem Cell Research & Therapy
» Transplantation Technologies & Research
   
Pharmaceutical Sciences
» Advances in Pharmacoepidemiology & Drug Safety
» Alcoholism and Drug Dependence
» Bioanalysis & Biomedicine
» Biochemical Pharmacology
» Bioequivalence & Bioavailability
» Cardiovascular Pharmacology: Open Access
» Clinical Pharmacology & Biopharmaceutics
» Developing Drugs
» Drug Designing
» Drug Metabolism & Toxicology
» Medicinal & Aromatic Plants
» Molecular Pharmaceutics & Organic Process Research
» Pharmaceutica Analytica Acta
» Pharmaceutical Regulatory Affairs
» Pharmacovigilance
» Vaccines & Vaccination
   
Chemistry
» Advanced Chemical Engineering
» Analytical & Bioanalytical Techniques
» Chemical Sciences Journal
» Chromatography & Separation Techniques
» Medicinal Chemistry
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» Natural Products Chemistry & Research
» Organic Chemistry
» Physical Chemistry & Biophysics
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» Thermodynamics & Catalysis
» Theoretical and Computational Chemistry:Open Access
   
Environmental
» Anthropology
» Aquaculture Research & Development
» Astrobiology & Outreach
» Biodiversity & Endangered Species
» Earth Science & Climatic Change
» Ecosystem & Ecography
» Environmental & Analytical Toxicology
» Fisheries & Livestock Production
»Fundamentals of Renewable Energy and Applications
» Geography & Natural Disasters
» Hydrology: Current Research
»Innovative Energy Policies
»International Journal of waste resources
» Oceanography-Open Access
» Petroleum & Environmental Biotechnology
» Pollution Effects & Control
» Poultry, Fisheries & Wildlife Sciences
   
Omics
» Data Mining in Genomics & Proteomics
»Ergonomics
» Glycomics & Lipidomics
» Health & Medical Informatics
»JBR Journal of Young Scientist
» Metabolomics:Open Access
» OMICS Journal of Radiology
» Pharmacogenomics & Pharmacoproteomics
» Phylogenetics & Evolutionary Biology
» Proteomics & Bioinformatics
» Transcriptomics
   
Life Sciences
» Advanced Techniques in Biology & Medicine
»Advancements in Genetic Engineering
»Advances in Crop Science and Technology
»Advances in Dairy Research
» Aging Science
» Agrotechnology
» Air & Water Borne Diseases
» Antivirals & Antiretrovirals
» Autism-Open Access
» Bacteriology & Parasitology
» Biochemistry & Physiology
» Biochemistry and Analytical Biochemistry
» Bioenergetics
» Bioengineering & Biomedical Science
» Biofertilizers & Biopesticides
» Biological Systems
» Biometrics & Biostatistics
»Biomimetics Biomaterials and Tissue Engineering
» Biomolecular Research & Therapeutics
» Bioremediation & Biodegradation
» Biosafety
» Biosafety & Health Education
» Biosensors & Bioelectronics
» Biosensors Journal
» Biotechnology & Biomaterials
» Bioterrorism & Biodefense
» Cell & Developmental Biology
» Current Synthetic & Systems Biology
» Entomology, Ornithology & Herpetology
» Enzyme Engineering
» Fermentation Technology
» Fertilization: In Vitro - IVF-Worldwide
» Fisheries and Aquaculture Journal
» Forest Research
» Fungal Genomics & Biology
» Gene Technology
» Glycobiology
» Horticulture
» Homeopathy & Ayurvedic Medicine
»International Journal of Swarm Intelligence and Evolutionary Computation
» Marine Science: Research & Development
» Membrane Science & Technology
» Microbial & Biochemical Technology
» Molecular Biology
» Molecular Imaging & Dynamics
» Nanomedicine & Biotherapeutic Discovery
» Nanomedicine & Nanotechnology
» Nutrition & Food Sciences
» Plant Pathology & Microbiology
» Primatology
» Probiotics & Health
» Rice Research: Open Access
» Single Cell Biology
» Sociology and Criminology-Open Access
» Tissue Science & Engineering
» Tropical Diseases
» Veterinary Science & Technology
» Vitamins & Minerals
»Virology & Mycology
   
Engineering
» Advances in Automobile Engineering
» Advances in Robotics & Automation
» Aeronautics & Aerospace Engineering
» Applied & Computational Mathematics
» Applied Mechanical Engineering
» Architectural Engineering Technology
» Astrophysics & Aerospace technology
» Biochips & Tissue Chips
» Bioprocessing & Biotechniques
» Chemical Engineering & Process Technology
» Civil & Environmental Engineering
» Computer Science & Systems Biology
» Electrical & Electronics
» Food Processing & Technology
» Geology and Geosciences
» Geophysics & Remote sensing
»Global Journal of Technology and Optimization
» Industrial Engineering & Management
» Information Technology & Software Engineering
» International Journal of Advance Innovations, Thoughts & Ideas
»International Journal of Sensor Networks and Data Communications
» Irrigation and Drainage Systems Engineering
»Generalized Lie Theory and Applications
» Lovotics
» Material Sciences & Engineering
»Physical Mathematics
» Powder Metallurgy & Mining
» Telecommunications System & Management
» Textile Science & Engineering
»Vortex Science and Technology
   
Medical Sciences
» Addiction Research & Therapy
» Allergy & Therapy
» Alternative & Integrative Medicine
» Alzheimers Disease & Parkinsonism
» Anaplastology
» Anatomy & Physiology
» Ancient Diseases & Preventive Remedies
» Andrology
» Arthritis
» Autacoids
»Automatic Control of Physiological State and Function
» Bioceramics Development and Applications
» Biology and Medicine
» Biomusical Engineering
» Blood & Lymph
» Blood Disorders & Transfusion
» Bone Marrow Research
» Brain Disorders & Therapy
» Cardiovascular Diseases & Diagnosis
» Cloning & Transgenesis
» Community Medicine & Health Education
» Dentistry
» Depression and Anxiety
» Diabetes & Metabolism
» Emergency Medicine
» Endocrinology & Metabolic Syndrome
» Epidemiology: Open Access
» Family Medicine & Medical Science Research
» Gastrointestinal & Digestive System
» General Medicine
» General Practice
» Genetic Syndromes & Gene Therapy
» Gynecology& Obstetrics
» Gerontology & Geriatrics Research
» Hematology & Thromboembolic Diseases
» Hair: Therapy & Transplantation
» Health Care : Current Reviews
» Hereditary Genetics
» Human Genetics & Embryology
» Hypertension-Open Access
» Infectious Diseases & Therapy
» International Journal of Genomic Medicine
» Internal Medicine
»International Journal of Biomedical Data Mining
» International Journal of Physical Medicine & Rehabilitation
»JBR Journal of Interdisciplinary Medicine and Dental Science
» Leukemia
» Liver
»Malaria Chemotherapy, Control & Elimination
» Medical & Surgical Urology
» Medical Diagnostic Methods
» Medical Microbiology & Diagnosis
» Metabolic Syndrome
» Molecular Biomarkers & Diagnosis
» Molecular and Genetic Medicine
» Mycobacterial Diseases
» Neonatal Biology
» Nephrology & Therapeutics
»Neuroinfectious Diseases
» Neurological Disorders
» Novel Physiotherapies
» Nuclear Medicine & Radiation Therapy
» Nursing & Care
» Nutritional Disorders & Therapy
» Obesity & Weight Loss Therapy
» Occupational Medicine & Health Affairs
» Oral Health and Dental Management
» Oral Hygiene & Health
» Orthopedic & Muscular System
» Osteoporosis & Physical Activity
» Otolaryngology
» Pain & Relief
» Palliative Care & Medicine
» Pancreatic Disorders & Therapy
» Pediatrics & Therapeutics
» Primary Health Care
» Psychological Abnormalities in Children
» Psychology & Psychotherapy
» Reproductive System & Sexual Disorders
»Research and Development
» Rheumatology: Current Research
» Sleep Disorders & Therapy
» Spine
» Sports Medicine & Doping Studies
» Steroids & Hormonal Science
» Surgery: Current Research
» Thyroid Disorders & Therapy
» Translational Medicine
» Trauma & Treatment
» Tropical Medicine and Surgery
» Vascular Medicine & surgery
» Women's Health Care
» Yoga & Physical Therapy
 
Management
» Accounting & Marketing
» Arabian Journal Business and Management Review
» Arts and Social Sciences Journal
» Business and Economics Journal
» Business and Financial Affairs
» Civil & Legal Sciences
» Defense Management
» Entrepreneurship & Organization Management
» Global Economics
» Hotel & Business Management
» Intellectual Property Rights: Open Access
»International Journal of Accounting Research
» International Journal of Economics & Management Sciences
» Mass Communication & Journalism
» political Sciences & public affairs
»Review of Public Adminstration and Management
» Socialomics
» Stock & Forex Trading
» Tourism & Hospitality
 
   Conferences
Upcoming Conferences
»3rd International Conference and Exhibition on Biosensors & Bioelectronics August 11-13, 2014 San Antonio, USA
»2nd World Associations Congress August 11-13, 2014 San Antonio, USA
»International Meeting on Space- Open Debate August 12-13, 2014 San Antonio, USA
»5th International Conference and Exhibition on Analytical & Bioanalytical Techniques August 18-20, 2014 Beijing, China
»2nd International Conference on Epidemiology and Evolutionary Genetics August 18-19, 2014 Beijing, China
»2nd International Conference and Exhibition on Pharmacognosy, Phytochemistry & Natural Products August 25-27, 2014 Beijing, China
»2nd International Conference and Exhibition on Traditional & Alternative Medicine August 25-26, 2014 Beijing, China
»2nd International Conference and Exhibition on Mechanical & Aerospace Engineering September 08-10, 2014 Philadelphia, USA
»2nd International Conference and Exhibition on Lasers, Optics & Photonics September 08-10, 2014 Philadelphia, USA
»3rd International Conference and Exhibition on Neurology & Therapeutics September 08-10, 2014 Philadelphia, USA
»4th International Conference on Pharmaceutical Regulatory Affairs September 08-10, 2014 Raleigh, USA
»2nd International Conference on Radiology and Imaging September 08-09, 2014 Raleigh, USA
»2nd International Conference on Genomics & PharmacogenomicsSeptember 08-09, 2014 Raleigh, USA
»2nd International Conference on Alzheimer's Disease and Dementia September 23-25, 2014 Valencia Convention Centre, Valencia, Spain
»3rd International Conference and Exhibition on Nutrition & Food Sciences September 23-25, 2014 Valencia, Spain
»3rd International Conference on Clinical Microbiology & Microbial Genomics September 24-26, 2014 Valencia, Spain
»3rd International Conference on Tissue Science & Regenerative Medicine September 24-26, 2014 Valencia, Spain
»4th International Conference on Vaccines & Vaccination September 24-26, 2014 Valencia, Spain
»3rd International Summit on GMP, GCP & Quality Control September 25-26, 2014 Valencia, Spain
»5th World Congress on Bioequivalence and Bioavailability: Pharmaceutical R&D Summit September 29-October 01, 2014 Baltimore, USA
»2nd International Conference on Hematology & Blood Disorders September 29-October 01, 2014 Baltimore, USA
»3rd International Conference and Exhibition on Clinical & Cellular Immunology September 29-October 01, 2014 Baltimore, USA
»3rd International Conference and Exhibition on Materials Science & Engineering October 06-08, 2014 San Antonio, USA
»3rd International Conference on Forensic Research and Technology October 06-08, 2014 San Antonio, USA
»4th World Congress on Virology October 06-08, 2014 San Antonio, USA
»2nd International Conference on Endocrinology October 20-22, 2014 Chicago, USA
»3rd 3rd International Summit on Toxicology & Applied Pharmacology October 20-22, 2014 Chicago, USA
»3rd 4th World Congress on Cancer Science and Therapy October 20-22, 2014 Chicago, USA
»3rd 3rd International Conference and Exhibition on Cell & Gene Therapy October 27-29, 2014 Las Vegas, USA
»2nd International Conference on HIV/AIDS, STDs & STIs October 27-29, 2014 Chicago, USA
»2nd World Congress on Petrochemistry and Chemical Engineering October 27-29, 2014 Las Vegas, USA
»3rd International Conference and Exhibition on Pharmacovigilance & Clinical Trials October 27-29, 2014 Hyderabad, India
»3rd International Conference and Exhibition on Biowaivers and Biosimilars October 27-29, 2014 Hyderabad, India
»3rd International Conference on Translational Medicine November 03-05, 2014 Las Vegas, USA
»2nd International Conference on Predictive, Preventive and Personalized Medicine & Molecular Diagnostics November 03-05, 2014 Las Vegas, USA
»5th International Conference on Diabetes & Metabolism November 03-05, 2014 Las Vegas, USA
»2nd International Congress on Bacteriology and Infectious Diseases November 17-19, 2014 Chicago, USA
»2nd International Conference on Nursing & Healthcare November 17-19, 2014 Chicago, USA
»3rd International Conference on Surgery and Anesthesia November 17-19, 2014 Chicago, USA
»4th International Conference on Nanotek & Expo December 01-03, 2014 San Francisco, USA
»3rd International Conference and Exhibition on Obesity & Weight Management December 01-03, 2014 San Francisco, USA
»2nd International Summit on Clinical PharmacyDecember 02-03, 2014 San Francisco, USA
»3rd International Conference on Medicinal Chemistry & Computer Aided Drug Designing December 08-10, 2014 San Francisco, USA
»3rd International Conference and Exhibition on Probiotics & Functional Foods December 08-10, 2014 San Francisco, USA
Previous Conferences Organized/Co-organized
»Health Informatics & Technology Conference July 28-29, 2014 Baltimore, USA
»3rd International Conference on Gastroenterology & Urology July 28-30, 2014 San Francisco, USA
»3rd International Conference on Earth Science & Climate Change July 28-30, 2014 San Francisco, USA
»3rd International Conference and Exhibition on Orthopedics & Rheumatology July 28-30, 2014 San Francisco, USA
»International Conference on Business Economics of Broadcasting Media & Film Industry July 28-30, 2014, Baltimore, USA
»4th International Conference on Proteomics & Bioinformatics August 04-06, 2014 Chicago, USA
»3rd International Conference and Exhibition on Addiction Research & Therapy August 04-06, 2014 Chicago, USA
»2nd International Conference on Integrative Biology Summit August 04-05, 2014 Chicago, USA
»3rd International Conference and Exhibition on Cosmetology & Trichology July 21-23, 2014 Las Vegas, USA
»3rd International Conference and Exhibition on Food Processing & Technology July 21-23, 2014 Las Vegas, USA
»2nd International Conference on Oceanography July 21-23 , 2014 Las Vegas USA
»4th International Conference on Clinical & Experimental Ophthalmology July 14-16, 2014 Baltimore, USA
»3rd International Conference & Exhibition on Biometrics & Biostatistics July 14-16, 2014 Baltimore, USA
»2nd International Conference and Exhibition on Physical Medicine & Rehabilitation July 14-16, 2014 Baltimore, USA
»International Conference on Geriatrics & Gerentology July 08-10, 2014 Chicago , USA
»International Conference on Women's Health, Gynecology & Obstetrics July 08-10, 2014 Chicago, USA
»International Conference on Computer Graphics and Media Design July 08-10, 2014 Chicago, USA
»4th International Conference on Clinical & Experimental Cardiology April 14-16, 2014 San Antonio, USA
»4th International Conference on Clinical & Experimental Dermatology April 14-16, 2014 San Antonio, USA
»3rd International Conference and Exhibition on Pathology April 14-16, 2014 San Antonio, USA
»5th International Conference on Biomarkers and Clinical Research April 15-17, 2014 University of Oxford, UK
»2nd International Conference on Business Economics and Management April 21-23, 2014 Dubai, UAE
»2nd International Conference on Dental and Oral Health April 21-23, 2014 Dubai, UAE
»4th World Congress on Cell Science & Stem Cell Research June 24-26, 2014 Valencia, Spain
»3rd International Conference on Biodiversity & Sustainable Energy Development June 24-26, 2014 Valencia, Spain
»3rd International Conference and Exhibition on Occupational Health & Safety June 24-25, 2014 Valencia, Spain
»5th World congress on Biotechnology June 25-27, 2014 Valencia, Spain
»3rd International Conference on Nephrology & Therapeutics June 26-27, 2014 Valencia, Spain
»2nd International Conference on Bioprocess and Engineering June 26-27, 2014 Valencia, Spain
»2nd International Conference on Dental and Oral Health April 21-23, 2014 Dubai, UAE
»5th International Conference on Biomarkers and Clinical Research April 15-17, 2014 University of Oxford, UK
»3rd International Conference and Exhibition on Pathology April 14-16, 2014 San Antonio, USA
»4th International Conference on Clinical & Experimental Dermatology April 14-16, 2014 San Antonio, USA
»4th International Conference on Clinical & Experimental Cardiology April 14-16, 2014 San Antonio, USA
»4th International Conference and Exhibition on Pharmaceutics & Novel Drug Delivery Systems March 24-26, 2014 San Antonio, USA
»3rd International Conference and Exhibition on Metabolomics & Systems Biology March 24-26, 2014 San Antonio, USA
»2nd International Conference on Agricultural & Horticultural Sciences, February 03-05, 2014 Hyderabad, India
»International Conference and Exhibition on Traditional & Alternative Medicine, December 09-11, 2013 Hyderabad, India
»2nd International Conference and Exhibition on Obesity & Weight Management, December 02-04, 2013 Las Vegas, USA
»International Conference on Nursing & Emergency Medicine, December 02-04, 2013 Las Vegas, USA
»3rd International Conference on Nanotek & Expo, December 02-04, 2013 Las Vegas, USA
»3rd World Congress on Virology, November, 20-22, 2013 Baltimore, USA
»International Congress on Bacteriology and Infectious diseases, November 20-22, 2013 Baltimore, USA
»3rd World Congress on Cell Science & Stem Cell Research, November 20-22, 2013 San Antonio, USA
»2nd International Conference and Exhibition on Pharmacovigilance & Clinical Trials, November 18-19, 2013 San Antonio, USA
»International Summit on Clinical Pharmacy & Dispensing, November 18-20, 2013 San Antonio, USA
»World Congress on Petrochemistry and Chemical Engineering, November 18-20, 2013 San Antonio, USA
»International Conference on Functional and Comparative Genomics & Pharmacogenomics, November 12-14, 2013 Chicago, USA
»2nd International Conference and Exhibition on Cosmetology & Trichology, November 12-14, 2013 Chicago, USA
»2nd International Summit on GMP, QA & QC, November 12-14, 2013 Chicago, USA
»2nd International Summit on GLP, GCP & Clinical Pharmacology, November 12-14, 2013 Chicago, USA
» International Conference on Fermentation Technology, Bioprocess and Cell Culture, October 28-30, 2013 Kansas City, USA
»2nd International Conference and Exhibition on Food Processing & Technology, October 28-30, 2013 Kansas City, USA
»International Conference on HIV/AIDS, STDs, & STIs, October 24-25, 2013 Orlando-FL, USA
»2nd International Conference and Exhibition on Cell & Gene Therapy, October 23-25, 2013 Orlando, USA
»2nd International Conference and Exhibition on Probiotics & Functional Foods, October 23-25, 2013 Orlando, USA
»International Conference and Exhibition on Pharmacognosy, Phytochemistry and Natural Products, October 21-23, 2013 Hyderabad, India
» 3rd World Congress on Cancer Science & Therapy, October 21-23, 2013 California, USA
»3rd International Conference on Pharmaceutical Regulatory Affairs, October 21-23, 2013 California, USA
»2nd International Conference on Clinical & Cellular Immunology, October 15-17, 2013 Las Vegas, USA
»2nd International Conference on Medicinal Chemistry & Computer Aided Drug Designing, October 15-17, 2013 Las Vegas, USA
»4th International Conference and Exhibition on Analytical & Bioanalytical Techniques, October 15-17, 2013 Las Vegas, USA
»2nd International Summit on Toxicology, October 07-09, 2013 Las Vegas, USA
»International Conference and Exhibition on Lasers, Optics & Photonics, October 7-9, 2013 San Antonio, USA
»2nd International Conference and Exhibition on Materials Science & Engineering, October 07-09, 2013 Las Vegas, USA
»2nd International Conference on Forensic Research & Technology, October 07-09, 2013 Las Vegas, USA
»International Conference and Exhibition on Biochemical & Molecular Engineering, October 07-08, 2013 San Antonio, USA
»International Conference on Psychology, Autism and Alzheimer's Disease, September 30-October 01, 2013 San Antonio, USA
»International Conference and Exhibition on Mechanical & Aerospace Engineering, September 30-October 02, 2013 San Antonio, USA
»2nd International Conference and Exhibition on Biowaivers & Biosimilars, September 23-25, 2013 Raleigh, USA
»4th World Congress on Biotechnology, September 23-25, 2013 Raliegh, USA
»International Conference on Hematology & Blood Disorders, September 23-25, 2013 Raliegh, USA
»2nd International Conference on Surgery and Anesthesia, September 16-18, 2013 Las Vegas, USA
»2nd International Conference on Clinical Microbiology & Microbial Genomics, September 16-18, 2013 Las Vegas, USA
» International Conference on Omics Studies, September 04-06, 2013 Orlando-FL, USA
»International Symposia on Entomology, September 04-06, 2013 Miami, USA
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Research Article Open Access
 
Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NF-Kappa B
1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA
2Department of Surgery, Texas Medical School at Houston, Houston, TX, USA
Phillip A. Letourneau1,2
, 1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA Tyler D. Menge1, 1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA Kathryn A. Wataha1, 1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA
2Department of Surgery, Texas Medical School at Houston, Houston, TX, USA
Charles E. Wade1,2
, 3Department of Pediatric Surgery, Texas Medical School at Houston, Houston, TX, USA Charles S.Cox Jr3, 1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA
2Department of Surgery, Texas Medical School at Houston, Houston, TX, USA
John B. Holcomb1,2
and
1Center for Translational Injury Research, Texas Medical School at Houston, Houston, TX, USA
2Department of Surgery, Texas Medical School at Houston, Houston, TX, USA
Shibani Pati1,2*
 
Corresponding Author : Shibani Pati, MD, PhD
Assistant Professor for Translational Injury Research
Department of Surgery University of Texas School of Medicine
Houston 6431 Fannin St. MSB 5.212 Houston, TX 77030
Tel: 713-500-5417
Fax: 713-512-7135
E-mail: Shibani.pati@uth.tmc.edu
 
Received July 06, 2011; Accepted July 23, 2011; Published July 25, 2011
 
Citation: Letourneau PA, Menge TD, Wataha KA, Wade CE, Cox CS Jr, et al. (2011) Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NFKappa B. J Tissue Sci Eng S3:001. doi: 10.4172/2157-7552.S3-001
 
Copyright: © 2011 Letourneau PA, et al. 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.
 
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Abstract
 

Bone marrow derived mesenchymal stem cells (MSCs) have been shown to demonstrate benefit in multiple disease models characterized by inflammation such as sepsis and acute lung injury. Mechanistically we hypothesized that MSCs exhibit these properties through inhibition of leukocyte activation and modulation of leukocyte-endothelial interactions; key interlinked processes involved in the deleterious effects of injury and inflammation. In this paper we found that MSCs co-cultured with a monocytoid line, U937, inhibit U937 binding to pulmonary endothelial cells (PECs) stimulated with the inflammatory cytokine TNF?. Furthermore, we show that these effects on functional adhesion are not due to changes in inflammatory adhesion molecule expression on U937s. No changes were found in CD62L, CD29, CD11b and CD18 expression on U937s co-cultured with MSCs. To determine if the effects of MSCs on leukocyte-endothelial interactions are due to the effects of MSCs on leukocyte activation, we investigated whether MSCs affect functional activation of the transcription factor NF-Kappa B. We found that MSCs significantly inhibit transcriptional activation of NF-kappa B in U937s. We also found that MSCs inhibit DNA binding of NF-kappa B subunits p50 and p65 to putative NF-kappa B DNA binding sites. Concomitant with a decrease in NF-kappa B activation was a significant increase in IL-10, an anti-inflammatory cytokine known to inhibit activation of NF-kappa B. Taken together, these findings show that MSCs have potent effects on leukocyte-endothelial interactions which may be due to the direct effects of MSCs on IL-10 and NF-kB. These findings suggest a potential therapeutic role for MSCs in diseases characterized by inflammation such as acute lung injury or multi-organ failure induced by traumatic injury.

 
Keywords
 
Acute lung injury; Progenitor cells; Inflammation
 
Abbreviations
 
MSCs: Mesenchymal stem cells; PECs: pulmonary endothelial cells; TBI: traumatic brain injury; ALI: Acute Lung Injury; ARDS; Acute Respiratory Distress Syndrome; FBS: Fetal bovine serum; PBS: phosphate buffered solution; PMA: phorbol myristate acetate
 
Introduction
 
Human mesenchymal stem cells (MSCs) are pluripotent stromal cells that have demonstrated benefits when infused in multiple preclinical models of inflammation and injury, such as myocardial infarction, stroke, traumatic brain injury and lung injury [1-5]. Many of these studies have shown potent systemic and local effects of MSCs. Our past work has shown that MSCs have potent stabilizing effects on the vascular endothelium when administered intravenously after traumatic brain injury (TBI) [5]. In these studies we found that MSCs inhibit blood brain barrier permeability when administered after TBI, which appears to be due to the presence of soluble factor(s) produced by MSC administration. In addition to these stabilizing effects on the vascular endothelium, a number of groups have shown that MSCs have potent anti-inflammatory effects in models of disease characterized by high levels of inflammation such as Acute Lung Injury and Acute Respiratory Distress Syndrome (ALI and ARDS) [6-8]. Utilizing a mouse model of sepsis and ALI, Nemeth et al. have shown that MSCs improved survival through modulation of the innate immune system, specifically by modulating the release of PGE2 from lung macrophages leading to increased production of the anti-inflammatory cytokine IL-10. [7,9]. MSCs have also been shown to modulate immunological responses via T-cell suppression [10-12]. To date MSCs have been investigated as a new therapeutic strategy for T cell-mediated diseases such as graft-versus-host disease (GVHD), Crohn’s disease, and the prevention of organ transplantation rejection [13]. In all of these disease contexts, limited numbers of MSCs have been observed to migrate to injured sites after systemic administration, but primarily most of the cells administered intravenously become lodged through a first-pass effect in the lung, liver or spleen regardless of the disease model [14,15]. Tissue-specific engraftment is not required and this aspect of MSC therapy in disease suggests a systemic effect of MSCs possibly produced by soluble factors.
 
Considering these data demonstrating an anti-inflammatory therapeutic effect of MSCs, we sought to find out more about their putative mechanisms of action. We hypothesized that MSCs regulate leukocyte activation and interaction with vascular endothelium, two processes that are highly interlinked and are key mediators of tissue injury in a number of disease states. To investigate these mechanisms of action, we designed in vitro experiments to study the effects of MSCs on leukocyte-endothelial interactions and leukocyte activation. Our findings do indeed suggest that MSCs inhibit inflammation and leukocyte-endothelial interactions through direct interactions with leukocytes.
 
Methods
 
Primary cells and cell lines: First passage human MSCs and pulmonary endothelial cells (PECs) were purchased from Lonza (Walkersville, MD). MSCs were cultured in MSC growth media (MSCGM, Lonza); PECs were maintained in EGM-2MV media from Lonza. MSCs were used at passage 3–7 for all experiments. U937, a monocytoid immortalized cell line, were obtained from ATCC (Bethesda, MD) and passaged in RPMI 1640 with 10% fetal bovine serum. All cell lines were maintained at 37oC and 5% CO2.
 
Leukocyte binding assay: PECs were grown on 96 well plates. Approximately, 1 x 104 cells/well were seeded and incubated at 37°C for 2 days or until confluent. U937s were serum starved for 24 hours in RPMI 1640 with 1% FBS before culture in the following groups: (1) U937s alone, and (2) U937s with MSCs (1:10 ratio) in direct contact. Adhesion molecule expression on PECs was stimulated by the addition of TNFα (50ng/ml). Co-cultures were incubated at 37°C for 16 hours. U937s were stimulated with phorbol myristate acetate (PMA, 200ng/ mL) for 20 minutes and then removed from co-cultures. Cells were labeled with Calcein-AM (Invitrogen, Carlsbad, CA), added to wells and allowed to adhere for one hour. Non-adherent cells were gently washed away in PBS and labeled cells that remained were quantified by fluorescent reading on the Biotek Synergy II (Biotek, Winooski, VT) at 490nm wavelength excitation and 520nm emission.
 
Flow cytometry: MSCs were characterized by flow cytometry as described previously [15]. U937s were characterized for surface adhesion markers and integrins by flow cytometry (BD Facs Calibur, BD Biosciences, San Jose, CA). U937 cells were cultured in 6 well plates for 24 hours in 2 groups: (1) U937s alone, and (2) U937s + MSCs (10:1 ratio with replicates of 4/group). The U937s were collected and subsequently treated with PMA (200ng/mL). After 20 minute incubation, the cells were collected and stained with fluorophore conjugated antibodies to CD11b, CD18, CD29, and CD62L (BD Biosciences). The cells were then analyzed on the BD LSR II flow cytometer (BD Biosciences). Expression of adhesion molecules was quantified by mean fluorescence intensity using the Flo-Jo Program software (Ashland, OR).
 
Nuclear NF-kappa B DNA binding activity: Nuclear extracts were prepared using the Nuclear Extract Kit from Active Motif (Carlsbad, CA) according to the manufacturer’s protocol. Protein concentration was determined by Bradford assay (Pierce Biochemicals). p50 and p65 DNA binding was quantified by Trans-AM NF-kappa B DNA binding ELISA from Active Motif (Carlsbad, CA), according to the manufacturer’s protocol. Nuclear extracts (2-5μg) were combined with a standardized biotinylated oligonucleotide and plated on to a streptavidin coated plate and incubated for one hour at room temperature. The plate was then washed three times with wash buffer and antibodies to NF-kappa B p65 and p50 subunits were added and incubated for one hour. HRP conjugated secondary antibody was then added and incubated for one hour. The plate was then developed and read on a 96 well plate spectrophotometer at 450nm with reference wavelength of 655nm.
 
Leukocyte NF-kappa B reporter gene assay: U937s were cotransfected with NF-kappa B cis-Reporter plasmid (1 μg/5 x 105 cells, Agilent Technologies, Santa Clara, CA) and CMV-Renilla luciferase plasmid (Promega Madison, WI) using the Amaxa electroporation system (Walkersville, MD), according to the manufacturer’s protocol. The cells were then cultured in RPMI-1640 with 10% FBS for 16 hours. U937s were then collected, pooled, counted and plated in 6 well plates either with or without MSCs. The cells were then incubated at 37˚C overnight. In the morning either lipopolysaccharide (LPS, 5μg/mL) or PBS (vehicle) was added to the dish. The cells were incubated again at 37˚C for 4 hours Figure 5. U937s were then collected, lysed and assayed according to the Promega (Madison, WI) Dual Luciferase Assay kit protocol.
 
Flow cytometric cytokine bead assay: U937s were cultured either alone in varying concentrations (1.5 x 105/mL, 3.75 x 105/mL, and 5 x 105), or with MSCs in varying ratios (1:2, 1:5 and 1:10) in 6 well plates for 12 hours. LPS (5μg/mL) or vehicle was then added to the plates to increase the baseline of NF-kappa B. After a four hour incubation the supernatants were collected. Cytokines (TNF, IL-1B, IL-6, IL-8, and IL- 10) were quantified by flow cytometry by following the manufacturer’s protocol for the human inflammatory cytokines bead assay kit (BD Biosciences Cat. #551811, Franklin Lakes, NJ).
 
Statistical analysis
 
Statistical analysis was performed using Microsoft Excel and Stata 11 (College Station, TX). Analysis was performed by Student’s t-test or one-way ANOVA with post-hoc Bonferroni correction, as needed.
 
Results
 
Mscs inhibit adhesion of U937s to pulmonary endothelial cells (pecs)
 
Leukocyte adhesion to endothelium is a critical component in the development of an inflammatory response to injury. Injured vasculature and activated leukocytes often upregulate expression of adhesion molecules that mediate binding between the two cell types. Multiple factors are responsible for the compromise of the endothelial barrier in injury. The lungs are often one of the most sensitive organs to inflammatory insults and compromise of the alveolar–capillary barrier is found early after the initial insult whether it is trauma or infection [16]. In addition to factors such as hypoxia and thrombin, inflammatory changes caused by cytokine and chemokine release, leukocyte adhesion, diapedesis and infiltration into the lungs have all been shown to contribute to the clinical severity and outcome in ALI/ARDS [3]. Taking this into account and the demonstrated predominance of IVadministered MSCs in the pulmonary vasculature, we chose to focus our studies upon the effects of MSCs on pulmonary endothelial cells (PECs). We sought to determine if MSCs modulate leukocyte binding to PECs stimulated with the inflammatory cytokine TNFα. In these studies, U937s were co-cultured with MSCs for 24 hours followed by stimulation with PMA, to stimulate calcium dependent binding (see schematic Figure 1A). PECs were treated with TNFα (10ng/ml) and binding of calcein-labeled cells to treated PECs was quantified by flourimetry Figure 1B. U937 cells are a monocytoid line that we have previously used to study leukocyte-endothelial adhesion [17,18]. Binding studies reveal that PMA stimulated U937-PEC binding and U937s co-cultured with MSCs demonstrate significantly decreased binding to PECs Figure 1B. Representative photographs of calceinlabelled U937s binding to PECs are depicted in Figure 1C. These results suggest that MSCs do indeed modulate leukocyte endothelial cell interactions.
 
MSCs do not affect U937 adhesion molecule expression
 
To determine if the binding differences noted were due to changes in adhesion molecule expression on the surface of the U937 cells, we analyzed the expression of CD62L, CD29, CD11b and CD18. These adhesion molecules and integrins mediate binding of leukocytes to endothelial cells and the surrounding extracellular matrix. CD11b is a component of LFA-1 which binds to ICAM-1 on endothelial cells and CD18 is a component of VLA-4 which binds to VCAM-1 on endothelial cells. In these studies, U937s were co-cultured with MSCs for 24 hours followed by stimulation with PMA for 20 minutes (See schematic in Figure 2A). Bright field microscopy in Figure 2B shows culture of the U937 cells alone and Figure 2C shows a representative co-culture of the two cell types where the adherent MSCs are apparent in the background with adherent U937s in the foreground. Flow cytometric analysis of the U937 cells after co-culture reveals that there are no significant differences in CD62L, CD29, CD11b and CD18 expression on the U937s. Figure 2D shows no significant changes in mean fluorescent intensity (n=4) for each of the representative markers. Figure 3 shows the lack of a change or shift in both groups in histogram form for each of the surface markers.
 
MSCs inhibit NF-kappa B DNA binding
 
Since we did not find any changes in leukocyte adhesion molecules with MSC treatment, we speculated that the potent differences found in leukocyte-endothelial binding may be due to an inhibition of leukocyte activation by MSCs. We chose to look at the effects of MSCs on the transcription factor NF-kappa B, a factor that has been extensively characterized in multiple disease models and has been shown to be involved in the regulation of inflammatory processes in disease [19-22]. Our past work has also shown that MSCs do in fact inhibit NFkappa B in Kaposi’s Sarcoma cells through modulation of the kinase Akt [15]. Using NF-kappa B activation as a surrogate readout for leukocyte activation, we found that nuclear extracts from U937s cocultured with MSCs demonstrate decreased NF-kappa B DNA binding activity determined by a DNA binding ELISA for p50 and p65 activity (see materials and methods). We found that NF-kappa B subunit p50 was significantly less active in nuclear extracts from U937s co-cultured with MSCs compared to nuclear extracts from U937s stimulated with PMA alone or U937s co-cultured with PECs as a control cell (0.63 ± 0.03 vs. 0.92 ± 0.14 and 0.63 ± 0.03 vs. 1.7 ± 0.3, respectively, p=0.05 Table 1). DNA binding of the p65 subunit of NF-kappa B was also significantly inhibited in U937s cultured with MSCs versus U937s stimulated with PMA or U937s co-cultured with PECs as a control cell (0.34 ± 0.06 vs. 0.55 ± 0.01 and 0.34 ± 0.06 vs. 0.793 ± 0.07, respectively, p<0.05, Table 1).
 
MSCs inhibit NF-kappa B transcriptional activation in U937s
 
Since our data suggest that MSCs inhibit activation of NF-kappa B in U937s, we sought to determine if our findings with NF-kappa B DNA binding would translate into a functional decrease in NF-kappa B mediated transcription. U937s were transfected with a reporter plasmid containing five NF-kappa B DNA binding sites upstream of a firefly luciferase reporter gene. All cells were also co-transfected with a control Renilla luciferase plasmid (see schematic Figure 4A) All U937 cells were simulated with LPS to increase the baseline of NFkappa B activation. Data from the reporter gene assay showed that LPS significantly increased nuclear NF-kappa B in U937 cells cultured alone (0.075 ± 0.002 vs. 0.003 ± 0.002, Figure 4B) U937 cells co-cultured with MSCs demonstrated decreased NF-kappa B transcriptional activation (0.050 ± 0.001, p<0.05 Figure 4B). Taken together these data do indeed suggest that MSCs inhibit NF-kappa B DNA binding and transcriptional activation, possibly providing an explanation for our noted effect of MSCs on leukocyte-endothelial interactions.
 
MSCs induce production of the anti-inflammatory cytokine IL-10
 
To better understand the mechanism of action of MSCs on the inhibition of NF-kappa B we sought to determine if MSCs modulate the expression of factors that may regulate NF-kappa B. IL-10 has been shown to be a key mediator and regulator of the anti-inflammatory effects of MSCs in multiple disease models [7]. IL-10 has also been clearly shown to regulate the activation of NF-kappa B in inflammatory cells [23]. Using a flow cytometric bead based assay, supernatants from co-cultured cells were analyzed for the production of IL-10. These studies reveal that the MSCs and unstimulated U937s do not produce detectable levels of IL-10, but when co-cultured, there is a significant increase in IL-10, (7.5 ± 0.53pg/mL vs. 0 ± 0 pg/mL, p<0.01, Figure 5), at both 1:2 and 1:5 ratios of MSCs to U937s. Taken together these data suggest that MSCs do increase production of the anti-inflammatory cytokine IL-10, which may contribute to the noted inhibition of NFkappa B and leukocyte endothelial interactions by MSCs.
 
Discussion
 
MSCs have demonstrated benefit in preclinical and clinical models of injury and inflammation. Clinical trials in multiple inflammatory conditions, including graft versus host disease (GVHD), and Crohn’s colitis, have demonstrated benefit, with many trials currently still underway [24]. In preclinical models of disease, MSCs have been shown to have potent effects in many models including ALI/ARDS, arthritis, acute hepatic failure, transplant, and diabetes [6,25-29]. Nemeth et al., have shown increased survival associated with MSC therapy in sepsis [7]. While this group and other investigators have examined the immunomodulatory effects of MSCs on inflammatory cells, few have investigated the effects in relation to the vascular endothelium. The stem cell niche and the interactions between cells created by the therapeutic delivery of MSCs are still wholly unknown. In this paper we sought to better understand a defined interaction between MSCs and inflammatory cells and how this interaction affects leukocyte-endothelial cell binding. Since MSCs are often administered for therapeutic purposes intravenously, one of the main cell types they come into contact with is inflammatory cells in the pulmonary vasculature [14]. Our studies aimed to recapitulate some of these interactions in vitro, to better understand mechanistically the antiinflammatory effects of MSCs.
 
In this study we show that inflammatory cells (U937s) co-cultured with MSCs display diminished potential to bind to pulmonary endothelial cells. Since we did not see large differences in leukocyte adhesion markers with MSC treatment, we hypothesized that MSCs may be affecting leukocyte-endothelial adhesion through inhibitory effects on leukocyte activation. To study this further, we utilized NFkappa B activation as a surrogate marker for leukocyte activation. We used two methods to show that MSCs decrease NF-kappa B activity in U937s; DNA binding and transcriptional activation by NF-kappa B. Co-culture of U937s with MSCs significantly diminished both measures of NF-kappa B activity. The role of NF-kappa B on inflammation and injury has been well documented throughout the years. Investigators in Germany isolated neutrophils from severely injured trauma patients and found significantly increased levels of NF-kappa B translocation to the nucleus compared to healthy volunteers [6,30]. Similarly, researchers utilizing an animal model of trauma and sepsis found increases in NF-kappa B activation in macrophages compared to sham. By inhibiting NF-kappa B, the authors were able to demonstrate decreased activation of alveolar macrophages and increased therapeutic benefit [20]. These data as a whole suggest an important role for NFkappa B in inflammatory disease outcome. It is of interest to note that regardless of changes in leukocyte adhesion markers, there are changes in MSC treated U937 that diminish binding. This data suggests that non-specific changes U937 may be taking place that alter adhesion such as changes in conformation or matrix interactions. Further study in this area is warranted.
 
To determine if IL-10 may play a role in the effects of MSCs on NF-kappa B, we investigated if IL-10 production was increased in our co-cultures. We indeed found that IL-10 was significantly increased in U937s by co-culture with MSCs suggesting a possible paracrine mechanism of action for the inhibition of NF-Kappa B in leukocytes by MSCs. Interestingly, the influence of MSCs on IL-10 production has been well documented in many models in vitro and in vivo and has been suggested by multiple groups to be a key source of the therapeutic benefit of MSCs [3,6,7,31]. A number of studies have also shown that MSCs function through soluble factors that produce a local paracrine and systemic effect [32-35]. There are many candidate factors that have been proposed as being critical to the therapeutic effects of MSCs. Our data in traumatic brain injury (TBI) support this view and have shown that intravenously delivered MSCs potently inhibit blood brain barrier permeability, without many cells present in the injured brain, suggesting the presence of soluble factors. These data and the data of others support the view that MSCs modulate inflammation and vascular stability through systemic, rather than local effects [14,36]. Previous work by our group has demonstrated that MSCs stabilize the endothelium and prevent disruption of the blood brain barrier through regulation of endothelial cadherin/beta-catenin signaling [5]. This effect combined with our data demonstrates a robust influence by MSCs on both the endothelium and the inflammatory response of blood leukocytes to decrease inflammatory capacity. Considering the multiple cell types involved in these interactions, the endothelial side of the “picture” is of great interest as well. Studies are currently underway to investigate the effects that MSCs have on endothelial cells and how this interaction affects leukocyte-endothelial cell interactions. Initial unpublished data from our group suggests that PECs treated with MSCs do decrease endothelial adhesion molecule expression and leukocyte binding.
 
In summary, we have shown that MSCs decrease leukocyteendothelial binding and NF-kappa B activation in inflammatory cells. Our data indicates that MSCs decrease leukocyte activation in response to stimulus which may be the explanation for the diminished binding to PECs. Decreases in NF-kappa B activity may be due to increased IL-10 production by monocytes and macrophages, resulting an overall decrease in the inflammatory state. MSCs remain an important area for translational study in treating the immunological response to injury, sepsis, and other inflammatory conditions.
 
Acknowledgements
 
The authors would like to thank Angela Beeler for her editorial support. The authors would also like to thank Scott Holmes for his help and expertise in graphics.
 
Funding and Support
 
This work was supported by funding from Mission Connect TIRR Foundation Houston, Texas and the National Institute of Health, T32GM008792-10.
 
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