Highlights

  • The NIMML team combines animal and computational modeling in an innovative product development pipeline
  • Successfully developed large animal models of infectious and immune-mediated diseases, including the development of pig models of IBD and H. pylori infection
  • Staff composed of researchers, veterinarians, immunologists, toxicologists and pathologists
  • Expertise in pre-clinical efficacy and safety testing of new drugs towards IND approval
  • Mechanism of action discovered for naturally occurring compounds and new chemical entities by combining knockout mice and bioinformatics approaches.
  • Integrated mouse colony management system and laboratory information management system for real-time data capture

Ongoing Efforts

The NIMML has over 15 years of experience in developing novel animal models of infectious, immune-mediated and metabolic chronic diseases. We have experience in mice, rats and pig models

TYPE II DIABETES ANIMAL MODELS

Diabetes mellitus type II is a chronic disease that is distinguished by insulin resistance and hyperglycemia (high concentrations of glucose in the blood) due to cells in the body not uptaking glucose.

Type II is a rapidly growing condition common in developed countries. Currently over 29 million Americans are diagnosed with Type II and out of all the cases of diabetes, Type II consists of 90% of the cases. Obesity, lifestyle, genetics, and previous medical conditions are the leading risk factors to Type II.

At NIMML we utilize animal models in order to simulate the proliferation of diabetic symptoms under conditions where potential biomolecular targets of medical interest can be manipulated and observed. In order to study these possible therapeutic targets and Type II NIMML has utilized the following mouse models.

  • The db/db transgenic mouse model
    • Cg-+Leprdb/+Leprdb/OlaHsd (db/db) strain mice are a model of obesity and diabetes caused by a deficiency in leptin receptor activity through gene knockdown. Due to this leptin deficiency, it causes the mice to become hyperphagic, obese and hyperglycemic. Obesity can be seen in mice after 5 weeks of age, followed quickly by hyperglycemia. The db/db mouse model insures quality genetically induced conditions in which diabetes symptoms can be accurately simulated.
  • Controlled diet mouse model
    • High-fat diet induced insulin resistance
      • C57BL/6J strain mice are fed a high fat isocaloric and isonitrogenous AIN-93G diet where calories obtained from fat are increased substantially (16% more kcal from fat). Obesity from the diet drives insulin resistance over time. As this model is induced by an environmental manipulation rather than genetic, it may be considered more accurately representing the common human condition.
    • Low-fat diet
      • C57BL/6J strain mice were fed purified isocaloric and isonitrogenous diets that represented a low fat modification of the AIN-93G diet commonly used for the growth, pregnancy, and lactation phases of mice.

Selected Publications

Activation of PPAR gamma and alpha by punicic acid ameliorates glucose tolerance and suppresses obesity-related inflammation.

http://www.ncbi.nlm.nih.gov/pubmed/19828904

Abscisic acid synergizes with rosiglitazone to improve glucose tolerance and down-modulate macrophage accumulation in adipose tissue: possible action of the cAMP/PKA/PPAR γ axis

http://www.ncbi.nlm.nih.gov/pubmed/20207056

Catalpic acid decreases abdominal fat deposition, improves glucose homeostasis and upregulates PPAR alpha expression in adipose tissue.

http://www.ncbi.nlm.nih.gov/pubmed/18778878

Dietary abscisic acid ameliorates glucose tolerance and obesity-related inflammation in db/db mice fed high-fat diets.

http://www.ncbi.nlm.nih.gov/pubmed/17000034

Data Figures

About

C. difficile experiments within NIMML follow a standard timeline of events. As mice are resistant to C. difficile infection with an intact intestinal flora, five days prior to infection with C. difficile (termed day -5), an antibiotic mixture (colistin, gentamycin, metronidazole, and vancomycin) is added to the water supply. Mice stay on the antibiotic water until day -3 of the timeline at which point mice will be placed back on standard water. On day -1, mice receive an intraperitoneal injection of clindamycin (32 mg/kg) to further remove any colonizing species. On day zero, mice are challenged with C. difficile through intragastric gavage. The strain VPI10463 is used for our studies. The model provides the opportunity to analyze an acute infection (through day 14) and a relapse period (through day 30).

The mouse model of C. difficile is able to be used to study a broad range of C. difficile related factors from the role of the microbiota to the balance of T cells to the activation of neutrophils to the formation of lesions. While the model requires the use of antibiotics to induce disease, the need for a reduced microbiota is not unlike the development of disease within humans, as most C. difficile infections are associated with antibiotic use. The model also provides the ability to study multiple events throughout the progression of disease from the initial colonization and response in early disease in days 1 through 3 and the peak of inflammation and disease on days 4 and 5 as well as the recovery phase and clearance of infection between days 8 and 10. Previously, we have utilized this model to establish systems level computational models of the host response to C. difficile, investigate the role of miRNA in the establishment of inflammation, and evaluate natural products for therapeutic efficacy.

Preclinical models for Infectious diseases: Respiratory pathogens

The NIMML has been working on Influenza models with several strains, being able to develop lung damage (i.e., epithelial necrosis, leucocyte infiltration), weight loss 10-20% and clinical disease. The virus strains we are using in mouse and pig challenge studies include:

  • H1N1 strain A/Puerto Rico/8/34 (PR8)
  • H3N2 strain A/Udorn/72
  • H1N1 strain A/California/07/2009

NIMML can provide data on:

  • Viral disease progression, weight loss
  • Histology and immunohistochemistry
  • Vaccine and therapeutic efficacy
  • Immunological assays and flow cytometry
  • Viral titers
  • LD50 studies

Preclinical models for Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is characterized by two major clinical manifestations: Crohn’s disease (CD) and ulcerative colitis (UC). CD can affect any regions of the gastrointestinal (GI) tract from mouth to anus. In contrast, UC causes acute colonic inflammation. The NIMML offers expertise and services in mouse models of IBD such as trinitrobenzene sulfonic acid (TNBS), dextran sodium sulfate (DSS)-induced colitis, pan-enteritis due to the deficiency of interleukin-10 (IL-10 KO) and CD4+ T cell-induced colitis in adoptive transfer models. Whereas TNBS and DSS-induced colitis represent the more acute and faster induction of colonic tissue damage, the IL-10 KO and the CD4+ T cell induced colitis represent chronic models of IBD. We have also developed a model of inflammation-driven colorectal cancer (CRC) following treatment of mice with azoxymethane and DSS. The NIMML provides expertise and resources in these four preclinical models with more than 10 years of experience and results published in top gastroenterology journals. NIMML can provide data on

  • Immunological changes at the gut mucosa and systemically
  • Gut histological and immunohistochemical changes
  • Transcriptomic analyses (RNAseq, ChipSep, miRNA)
  • Pre-clinical efficacy, tolerability and weight loss
  • Cellular and molecular profiling

Nutritional Immunology and Molecular Medicine Preclinical Studies

The NIMML pre-clinical studies have an added translational value by the incorporation of pig models of infectious and immune-mediated diseases, including inflammatory bowel disease (IBD) and infections caused by enteric or respiratory pathogens. As opposed to mice, pigs lend a physiological composition more comparable to humans, specifically the mucosal immune system, of the pig more closely resembles that of human and are thus an ideal model for studying the complexity of the human immune system and how it responds to inflammation, infection, and injury. Preclinical studies using pigs lay the groundwork for an informed design of human clinical trials.

Our Facilities and Animals

Our AAALAC-accredited swine facility consists of 16 separate experimental housing rooms with capacities of 8 pigs each. A fully equipped surgical room is located in the building for onsite collection of specimens. Supervisors, students and animal technicians are able to provide superior husbandry for pigs spanning all ages of the life cycle. The majority of our projects use neonatal to weaned pigs. Gnobiotic swine litters are also available. Procedures routinely performed in the swine studies include venipuncture, injections, immunizations, and intubation. All procedures and experiments are approved by the Institutional Animal Care and Use Committee (IACUC). Our facility is AAALAC-accredited and can be used for biosafety level 1 (ABSL1) and ABSL2 studies. Moreover, the studies can be run as Good Laboratory Practices (GLP), thereby fulfilling the requirements of regulatory agencies for IND packages. Active monitoring post-challenge is performed to ensure the well-being of the animals as well as provide an assessment of the disease activity progression throughout the duration of the experiment. Peripheral blood collection is performed on a regular basis as a means of analyzing the time course of an infection. Execution of specific expected goals is met with superior efficiency in a timely manner while maintaining detailed attention to our animals and data.

Neonatal Model

Extensive research has shown the similarities in human neonates compared to swine neonates regarding neonatal nutrition and digestion as well as the immunoregulatory system thus rendering this type of model preferable over mice or other rodent species for pre-clinical trials. Changes in neonatal pigs represent rapid intestinal growth and functions surrounding nutrient absorption and immune functions. When studying the effects of diseases such as Enteroaggregative E. coli or rotovirus which are particular to neonates, the demand to use a model that is more representative of the chronic infections is necessary. Swine have a gestation length of 113-115 days and synchronization techniques allow for timed parturition and project set-up. Piglets used for neonatal projects are removed from the mother immediately at birth to be placed into highly regulated state of the art facilities uniquely designed for the piglets. Our facilities are able to provide specialty care for early weaned neonates and maintain projects for long term. Piglets have twenty-four hour access to milk by means of our custom designed bottles which are continuously checked by trained personnel. Piglets are housed in their own incubator for intensive monitoring and thermoregulation control as well as individual feeding. Specialty care throughout the duration of their lives is under strict control of IACUC approved protocols.

Selected Publications

  • Differential requirements for proliferation of CD4+ and gammadelta+ T cells to spirochetal antigens. Hontecillas R, Bassaganya-Riera J. Cell Immunol. 2003 Jul;224(1):38-46. [PubMed]
  • Conjugated linoleic acid ameliorates viral infectivity in a pig model of virally induced immunosuppression. Bassaganya-Riera J, Pogranichniy RM, Jobgen SC, Halbur PG, Yoon KJ, O’Shea M, Mohede I, Hontecillas R. J Nutr. 2003 Oct;133(10):3204-]14. [PubMed]
  • Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine CD8(+) lymphocytes. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ. J Nutr. 2001 Sep;131(9):2370-7. [PubMed]
  • Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. Bassaganya-Riera J, Hontecillas-Magarzo R, Bregendahl K, Wannemuehler MJ, Zimmerman DR. J Anim Sci. 2001 Mar;79(3):714-21. [PubMed]

Gut Inflammation Studies

Inflammatory bowel disease (IBD) is a chronic reoccurring inflammatory illness affecting millions of people worldwide. IBD has two clinical manifestations: Crohn’s disease and ulcerative colitis. The NIMML developed an acute model of colitis in pigs by intragastric administration of dextran sulfate sodium (DSS) which causes epithelial erosion and subsequent leucocytic infiltration. Another chemical irritant which induces a slightly more severe IBD is 2,4,6-trinitrobenzene sulphonic acid (TNBS) which induces T helper 1 (Th1) and Th17 responses in the colonic mucosa. The NIMML can implement pig projects of DSS, TNBS or bacterial-induced colitis to measure the anti-inflammatory efficacy of new IBD treatments. Gut Inflammation Studies Provide Data for:

  • Biomarker discovery
  • Therapeutic target discovery
  • Bioinformatics analyses
  • Immunological assays
  • Histopathology services

Selected Publications

Infectious Disease Studies: Gastrointestinal Pathogens

    The NIMML has a primary focus on studying the mucosal immune responses to gastrointestinal and respiratory pathogens. Likewise, pre-clinical studies involving infectious disease also provide valuable mechanistic insights on efficacy and safety of vaccines and immune therapeutics. Our projects produce extensive results which can be implemented directly into our modeling efforts for calibration and fitting of model parameters. This approach allows us to begin inferring methods which are more predictive in studying a complex biological system. Risks for infection caused by gastrointestinal pathogens increases for people who travel internationally, live impoverished under malnourished diets, are immunocompromised, or consume the pathogen through contaminated food resulting in a food borne illness. Initial infection usually leads to gastrointestinal discomfort and subsequent diarrhea. When challenged with these pathogens and a persistent chronic illness occurs, painful ulcers form in the gastric (H. pylori) and intestinal tracts due to prolonged inflammation and the necrosis of tissue. Pre-clinical models of these pathogens are vital in the development of therapeutics which will translate into the development human clinical trials. The primary gastrointestinal infectious diseases studied by NIMML under the MIEP program include infections caused by Helicobacter pylori, Clostridium difficile, and Enteroaggregative Escherichia coli (EAEC) although other models can be easily developed.
    Specific strains used for infectious disease models:
    • Enteroaggregative Escherichia coli(EAEC)
      • JM221 Strain
      • 042 Strain

    • Helicobacter pylori

      • European 26695 strain
      • African J99 strain
      • SS1 strain

    • Clostridium difficile

      • UVA13 strain
      • VPI 11186 strain
      • 10463 strain

    The NIMML is a fully integrated translational effort with capabilities of performing a wide variety of validated immunological assays in pre-clinical and clinical settings as GLP or non-GLP. Experimental variation is tightly controlled through the refinement of protocols for techniques thus generating reliable quality data and our laboratory information management and integration systems.

    Selected Publications

Infectious Disease Studies: Respiratory Pathogens

The seasonal flu is a highly contagious illness which affects between 5-20% of people annually in the US. Due to the mutagenic characteristic of the orthomyxovirus, new strains appear each year amounting more than 200,000 hospitalizations due to infection and respiratory complications. The NIMML primarily studies influenza A virus. This genus has the ability to cause detrimental pandemics in a short period of time since it can be transmitted among different species. Specific strains in the NIMML include: H1N1 strain A/Pureto Rico/8/34 (PRS), H3N2 strain A/Udorn/72, and the H1N1 strain A/California/07/2009.

Our projects provide reliable data for:

  • Clinical weight loss
  • Viral disease progression
  • Lung histology and immunohistochemistry
  • Immunological Assays and flow cytometry

In addition, the NIMML has received IACUC approval to conduct LD50 studies for influenza projects.

Selected Publications

Swine as a Translational Model

The NIMML has the ability to focus on diseases on a multitude of levels. Our projects begin using mouse models and as the data becomes more comprehensive, pigs provide the intermediate step prior to clinical trials. Our pig models have provided data that is critical in understanding the immune response to colitis, IBD, enteric pathogens, and respiratory infections. We continue to use the pig model to facilitate the development of projects which can assist in computational modeling components of the laboratory as well as potentiate the translation of research into human patients. Our streamlined facilities allow us to work with collaborators to collect data and allow for personnel to focus on their specializations. By using students trained in animal sciences and other degrees which provide education encompassing animal models for scientific research, our team is able to work together to make novel advancements in a broad range of scientific topics with efficiency and accuracy.

Preclinical services

The Nutritional Immunology and Molecular Medicine Laboratory offers a broad array of preclinical services in mouse, rats, hamsters and pigs for product testing, mechanism of action validation studies, and hypothesis–driven and hypothesis-generating in vivo experimentation. All our preclinical animal models are set up in Virginia Tech animal facilities and are approved by the Virginia Tech Institutional Animal Care and Use Committee (IACUC). The mice are housed in a state-of-art facility that can accommodate large projects as well as both animal biosafety level (ABL)-1 and ABL-2 experiments. These services will be provided as a part of a sponsored research agreement with Virginia Tech.

Mouse Models

Colitis (DSS, adoptive transfer of CD4+ T cells, IL-10 ko, TNBS, bacterial induced) Influenza infection Clostridium difficile infection Enteroaggregative Escherichia coli infection Helicobacter pylori infection Colorectal cancer Type 2 diabetes and obesity (db/db, diet-induced obesity, KK-Ay, ob/ob) Lupus Type 1 diabetes (NOD, STZ) Atherosclerosis (ApoE)

Pig Models

Colitis (Bacterial-induced, DSS, TNBS) Influenza infection Helicobacter pylori infection Neonatal models Radiation models

Rat Models

Type 1 diabetes Type 2 diabetes Safety and toxicity assessment PK/PD, ADME

Preclinical models for Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is characterized by two major clinical manifestations: Crohn’s disease (CD) and ulcerative colitis (UC). CD can affect any regions of the gastrointestinal (GI) tract from mouth to anus. In contrast, UC causes acute colonic inflammation. The NIMML offers expertise and services in mouse models of IBD such as trinitrobenzene sulfonic acid (TNBS), dextran sodium sulfate (DSS)-induced colitis, pan-enteritis due to the deficiency of interleukin-10 (IL-10 KO) and CD4+ T cell-induced colitis in adoptive transfer models. Whereas TNBS and DSS-induced colitis represent the more acute and faster induction of colonic tissue damage, the IL-10 KO and the CD4+ T cell induced colitis represent chronic models of IBD. We have also developed a model of inflammation-driven colorectal cancer (CRC) following treatment of mice with azoxymethane and DSS. The NIMML provides expertise and resources in these four preclinical models with more than 10 years of experience and results published in top gastroenterology journals. NIMML can provide data on

  • Immunological changes at the gut mucosa and systemically
  • Gut histological and immunohistochemical changes
  • Transcriptomic analyses (RNAseq, ChipSep, miRNA)
  • Pre-clinical efficacy, tolerability and weight loss
  • Cellular and molecular profiling

Preclinical models for Infectious diseases: Respiratory pathogens

The NIMML has been working on Influenza models with several strains, being able to develop lung damage (i.e., epithelial necrosis, leucocyte infiltration), weight loss 10-20% and clinical disease. The virus strains we are using in mouse and pig challenge studies include:

  • H1N1 strain A/Puerto Rico/8/34 (PR8)
  • H3N2 strain A/Udorn/72
  • H1N1 strain A/California/07/2009

NIMML can provide data on:

  • Viral disease progression, weight loss
  • Histology and immunohistochemistry
  • Vaccine and therapeutic efficacy
  • Immunological assays and flow cytometry
  • Viral titers
  • LD50 studies

Preclinical models of Infectious diseases: Gastrointestinal pathogens

Yearly outbreaks of several pathotypes of E. coli, Salmonella, and other enteric pathogens incur medical costs, lost productivity and even premature death, with total expenses exceeding $6.9 billion per year. A closer comprehension of the mechanisms of action underlying immune responses to enteric pathogens will lead to the development of more efficacious vaccines and immunotherapeutics. Through the Modeling Immunity to Enteric Pathogens program, NIMML has build capabilities in modeling gut enteric infections. NIMML can reproduce and validate the following infectious disease models:

  • Enteroaggregative Escherichia coli(EAEC)
    • JM221 strain
    • 042 strain

  • Helicobacter pylori

    • European 26695 strain
    • African J99 strain
    • SS1 strain

  • Clostridium difficile

    • UVA13 strain
    • VPI 11186 strain
    • 10463 strain

Preclinical models to validate computational model approaches

The NIMML works in the interface between the experimental and modeling/simulation approaches to study the host responses to gut and respiratory pathogens. The modeling process requires strong crosstalk with the experimental side: model calibration and validation. Each model needs to be calibrated with experimental data to ensure correct fitting and the correct assignment on numerical values for the parameters in differential equations. Afterwards, when the model is fully calibrated with experimental data, a new window to experimental design is opened: model validation. Resulting in model-aided, hypothesis-driven experimental validation, positioning computational modeling as a effective and advanced tool for immunology research and discovery. The NIMML has expertise in developing new designs depending on the needs of the customer. We are currently working in several validation studies for mathematical models under the MIEP program and we are flexible to design new models with creation of knockouts with the zinc finger technology. This validation approach will accelerate the whole process into a final publication, showing how computational method can validate biological processes.

Selected References

  1. Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease Bassaganya-Riera J, Reynolds K, Martino-Catt S, Cui Y, Hennighausen L, Gonzalez F, Rohrer J, Benninghoff AU, Hontecillas R. Gastroenterology. 2004 Sep;127(3):777-91. [PubMed]
  2. Peroxisome proliferator-activated receptor gamma is required for regulatory CD4+ T cell-mediated protection against colitis Hontecillas R, Bassaganya-Riera J. J Immunol. 2007 Mar 1;178(5):2940-9. [PubMed]
  3. T cell PPARγ is required for the anti-inflammatory efficacy of abscisic acid against experimental IBD. Guri AJ, Evans NP, Hontecillas R, Bassaganya-Riera J. J Nutr Biochem. 2011 Sep;22(9):812-9. Epub 2010 Dec 15. [PubMed]
  4. Immunoregulatory actions of epithelial cell PPAR gamma at the colonic mucosa of mice with experimental inflammatory bowel disease. Mohapatra SK, Guri AJ, Climent M, Vives C, Carbo A, Horne WT, Hontecillas R, Bassaganya-Riera J. PLoS One. 2010 Apr 20;5(4):e10215. [PubMed]
  5. Immunoregulatory mechanisms of macrophage PPAR-γ in mice with experimental inflammatory bowel disease. Hontecillas R, Horne WT, Climent M, Guri AJ, Evans C, Zhang Y, Sobral BW, Bassaganya-Riera J. Mucosal Immunol. 2011 May;4(3):304-13. Epub 2010 Nov 10. [PubMed]
  6. The role of T cell PPAR gamma in mice with experimental inflammatory bowel disease. Guri AJ, Mohapatra SK, Horne WT 2nd, Hontecillas R, Bassaganya-Riera J. BMC Gastroenterol. 2010 Jun 10;10:60. [PubMed] 7. Dietary α-eleostearic acid ameliorates experimental inflammatory bowel disease in mice by activating peroxisome proliferator-activated receptor-γ. Lewis SN, Brannan L, Guri AJ, Lu P, Hontecillas R, Bassaganya-Riera J, Bevan DR. PLoS One. 2011;6(8):e24031. Epub 2011 Aug 31. [PubMed]