ABSTRACT
BACKGROUND: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. METHODS: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. RESULTS: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. CONCLUSION: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.
Subject(s)
Antigens, Neoplasm/immunology , CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/transplantation , Immunotherapy, Adoptive , Neoplasm Proteins/immunology , Neoplasms/therapy , Receptors, Chimeric Antigen/immunology , A549 Cells , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , CD8 Antigens/genetics , CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Coculture Techniques , Cytotoxicity, Immunologic , Female , HEK293 Cells , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , Humans , Immunodominant Epitopes , K562 Cells , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/metabolism , Phenotype , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
Dose selection for the 6-month rasH2 mouse carcinogenicity studies depends heavily on the maximum tolerated dose (MTD) obtained from 1-month range-finding studies. A retrospective evaluation of range-finding studies and pivotal 6 month rasH2 mouse studies for 11 compounds demonstrated that the MTD based on at least a 10% decrease in body weight gain, mortality, and target organ toxicity in range-finding studies appropriately identified high doses for pivotal studies for 8 of 11 compounds. Two of the selected high doses were based on decreased body weight gain alone, while 7 were based on mortality at higher doses in shorter duration range-finding studies. High-dose selection was based on the maximum feasible dose for one study. The Center for Drug Evaluation and Research, U.S. Food and Drug Administration Executive Carcinogenicity Assessment Committee often suggested different doses than those proposed by the sponsor. High mortality was observed in only one pivotal study and the high dose was lowered during the course of that study.
Subject(s)
Carcinogenicity Tests/methods , Drug Evaluation, Preclinical/methods , Animals , Female , MaleABSTRACT
Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment.
Subject(s)
Carcinogens/toxicity , Nitriles/toxicity , Pyrazoles/toxicity , Reverse Transcriptase Inhibitors/toxicity , Animals , Body Weight/drug effects , Carcinogenicity Tests , Dose-Response Relationship, Drug , Eating/drug effects , Female , Kaplan-Meier Estimate , Kidney Neoplasms/chemically induced , Kidney Neoplasms/pathology , Liver Neoplasms/chemically induced , Liver Neoplasms/pathology , Male , Nitriles/pharmacokinetics , Pyrazoles/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Inhibitors/pharmacokinetics , Survival Analysis , Thyroid Neoplasms/chemically induced , Thyroid Neoplasms/pathology , Urinalysis , Urinary Bladder Neoplasms/chemically induced , Urinary Bladder Neoplasms/pathologyABSTRACT
Thionamides such as propylthiouracil (PTU) and methimazole (MMI) have been used for more than 50 years to treat the more common causes of thyrotoxicosis/hyperthyroidism such as Graves' disease. Serious adverse effects associated with thionamides in humans include idiosyncratic liver damage, agranulocytosis, aplastic anemia, and vasculitis. Both prospective and retrospective clinical studies with these drugs have failed to identify predictive biomarker for these adverse effects. To assess whether rat is a good model for predicting drug-related adverse events in the liver and in the bone marrow, we conducted a comprehensive study in male rats with multiple doses of PTU and MMI. As expected, euthyroid animals became hypothyroid along with several secondary changes associated with hypothyroidism. There were slight reductions in red blood cell parameters along with some marginal effects on the bone marrow elements. However, there was no evidence of significant neutropenia and liver injury in both PTU-treated and MMI-treated cohorts. MMI-related effects were noted in the seminiferous tubules of the testes. Overall, 1-month daily treatment of euthyroid rats with PTU or MMI resulted in hypothyroidism, minor bone marrow effects, and several secondary effects associated with hypothyroidism, but without any evidence of adverse effects reported in humans including liver injury and agranulocytosis.
Subject(s)
Methimazole/toxicity , Propylthiouracil/toxicity , Testis/drug effects , Thyroid Gland/drug effects , Animals , Male , Methimazole/administration & dosage , Methimazole/blood , Methimazole/pharmacokinetics , Propylthiouracil/administration & dosage , Propylthiouracil/blood , Propylthiouracil/pharmacokinetics , Rats , Rats, Wistar , Testis/chemistry , Testis/pathology , Thyroid Gland/chemistry , Thyroid Gland/pathology , Toxicity TestsABSTRACT
International regulatory and pharmaceutical industry scientists are discussing revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S1 guidance on rodent carcinogenicity assessment of small molecule pharmaceuticals. A weight-of-evidence approach is proposed to determine the need for rodent carcinogenicity studies. For compounds with high human cancer risk, the product may be labeled appropriately without conducting rodent carcinogenicity studies. For compounds with minimal cancer risk, only a 6-month transgenic mouse study (rasH2 mouse or p53+/- mouse) or a 2-year mouse study would be needed. If rodent carcinogenicity testing may add significant value to cancer risk assessment, a 2-year rat study and either a 6-month transgenic mouse or a 2-year mouse study is appropriate. In many cases, therefore, one rodent carcinogenicity study could be sufficient. The rasH2 model predicts neoplastic findings relevant to human cancer risk assessment as well as 2-year rodent models, produces fewer irrelevant neoplastic outcomes, and often will be preferable to a 2-year rodent study. Before revising ICH S1 guidance, a prospective evaluation will be conducted to test the proposed weight-of-evidence approach. This evaluation offers an opportunity for a secondary analysis comparing the value of alternative mouse models and 2-year rodent studies in the proposed ICH S1 weight-of-evidence approach for human cancer risk assessment.
Subject(s)
Carcinogenicity Tests/standards , Disease Models, Animal , Neoplasms/drug therapy , Risk Assessment , Animals , Drug Evaluation, Preclinical , Drug Industry , Drug-Related Side Effects and Adverse Reactions , Humans , Mice , Mice, TransgenicABSTRACT
A factor limiting widespread use of the transgenic rasH2 mouse model for carcinogenicity testing of pharmaceuticals is the paucity of published data on actual drug candidates in rasH2 mice. This report addresses this gap by highlighting rasH2 mouse study data for 10 pharmaceutical candidates. These results were compared with findings in the 2-year studies in Sprague-Dawley rats for the same 10 compounds. In the 6-month rasH2 studies, only 2 of the 10 compounds tested positive for carcinogenicity and these correlated with positive findings in the companion 2-year rat studies. One compound, sunitinib, produced gastroduodenal carcinoma in both sexes and increased hemangiosarcoma in spleen and uterus in female rasH2 mice; in rats it produced gastroduodenal carcinoma and increased pheochromocytoma (males only). The second compound, bazedoxifene, produced ovarian granulosa cell neoplasms in rasH2 mice and rats, and renal tubular neoplasms associated with increased chronic progressive nephropathy only in rats. The higher percentage of carcinogenicity positive rat bioassays could be attributed to rat-specific phenomena with little or low relevance to man. Thus, this article confirms previous reports that rasH2 mice develop rodent-specific neoplasms less frequently than rats and positive findings, when present, are accompanied by similar positive results in the rat.
Subject(s)
Carcinogenicity Tests/methods , Models, Animal , Neoplasms, Experimental/chemically induced , Animals , Body Weight/drug effects , Carcinogenicity Tests/standards , Cell Proliferation/drug effects , Female , Genes, ras , Indoles/toxicity , Male , Mice , Mice, Transgenic , Neoplasms, Experimental/genetics , Pyrroles/toxicity , Rats , Rats, Sprague-Dawley , SunitinibABSTRACT
Overexpression of TMPRSS4, a cell surface-associated transmembrane serine protease, has been reported in pancreatic, colorectal and thyroid cancers, and has been implicated in tumor cell migration and metastasis. Few reports have investigated both TMPRSS4 gene expression levels and the protein products. In this study, quantitative RT-PCR and protein staining were used to assess TMPRSS4 expression in primary non-small cell lung carcinoma (NSCLC) tissues and in lung tumor cell lines. At the transcriptional level, TMPRSS4 message was significantly elevated in the majority of human squamous cell and adenocarcinomas compared with normal lung tissues. Staining of over 100 NSCLC primary tumor and normal specimens with rabbit polyclonal anti-TMPRSS4 antibodies confirmed expression at the protein level in both squamous cell and adenocarcinomas with little or no staining in normal lung tissues. Human lung tumor cell lines expressed varying levels of TMPRSS4 mRNA in vitro. Interestingly, tumor cell lines with high levels of TMPRSS4 mRNA failed to show detectable TMPRSS4 protein by either immunoblotting or flow cytometry. However, protein levels were increased under hypoxic culture conditions suggesting that hypoxia within the tumor microenvironment may upregulate TMPRSS4 protein expression in vivo. This was supported by the observation of TMPRSS4 protein in xenograft tumors derived from the cell lines. In addition, staining of human squamous cell carcinoma samples for carbonic anhydrase IX (CAIX), a hypoxia marker, showed TMPRSS4 positive cells adjacent to CAIX positive cells. Overall, these results indicate that the cancer-associated TMPRSS4 protein is overexpressed in NSCLC and may represent a potential therapeutic target.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Cell Hypoxia , Lung Neoplasms/metabolism , Membrane Proteins/biosynthesis , Serine Endopeptidases/biosynthesis , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Animals , Antigens, Neoplasm/analysis , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carbonic Anhydrase IX , Carbonic Anhydrases/analysis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Lung/metabolism , Lung Neoplasms/genetics , Membrane Proteins/genetics , Mice , Mice, Nude , RNA, Messenger/biosynthesis , Serine Endopeptidases/genetics , Transcription, Genetic , Tumor Microenvironment , Up-RegulationABSTRACT
Alternate transgenic mouse models are accepted as replacements for the standard carcinogenicity mouse bioassay by regulatory agencies with a companion 2-year rat bioassay. The slower rate of industry acceptance of these shorter transgenic mouse cancer bioassays has been due to lack of historical data and diagnostic criteria, and the use of nonstandardized terminologies in published data. To address these issues, especially that of generating a large historical database, a retrospective analysis of the spontaneous tumor incidences in rasH2 mice from internally sponsored 6-month carcinogenicity studies was compared to the published literature. Incidences of common spontaneous tumors (incidences > 1%) observed in these studies were lung bronchiolo-alveolar adenomas (mean 3.9-9.9%; range 0-18%), lung bronchiolo-alveolar adenocarcinomas (mean 1.4-2.4%; range 0-5%), splenic hemangiosarcomas (mean 3.0-3.9%; range 0-17%), cutaneous squamous cell papillomas (mean 1.1-1.2%; range 0-4%), Harderian gland adenoma (mean 0.8-1.2%; range 0-4%), and hepatocellular adenomas (mean 1.8%; 0-9% in males only). The remarkable similarity in the tumor incidences in multiple rasH2 studies over a decade and the observed stability of the inserted human gene are important indicators of the minimal drift in this model. Overall, the historical control data for spontaneous neoplasms should assist in the interpretation of future rasH2 mouse studies.
Subject(s)
Disease Models, Animal , Genes, ras , Neoplasms, Experimental/genetics , Animals , Body Weight , Carcinogenicity Tests , Female , Male , Mice , Mice, Transgenic , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/pathology , Phenotype , Survival AnalysisABSTRACT
Despite an increased understanding of the molecular pathogenesis of colorectal cancer (CRC) during the past two decades, reliable and robust biomarkers to enable screening, surveillance, and primary prevention of this disease are lacking. CRC diagnosis and therapy remain dependent upon descriptive classification and staging systems, based primarily on morphology and histology. The traditional approach of understanding complex biological systems by studying smaller, discrete units of the whole system has been less fruitful for understanding complex diseases. The implicit assumption of traditional methods, that a single or even only a few factors, play a dominant role in a complex disease might be inadequate when studying multifactorial diseases such as cancer. The burgeoning field of systems biology adopts a holistic approach, wherein the integration of individual parts of the system is sought. The cornerstone of a systems biology approach has been the development of a variety of high-throughput "omics" sciences, including genomics, transcriptomics, proteomics, and metabolomics. This review will focus on the "omics" literature in the field of sporadic human CRC and present examples of how a systems approach has been extremely useful in understanding concepts that would have been difficult to develop using traditional methods.
Subject(s)
Colorectal Neoplasms/genetics , Systems Biology/methods , Biomarkers/analysis , Genomics/methods , Humans , Metabolomics , Proteomics/methodsABSTRACT
Genetic mutations resulting in obesity and type 2 diabetes mellitus (T2D) are described for both inbred and outbred mice. However, no known mouse model completely recapitulates human T2D and its comorbidities. We identified a cohort of obese, male, outbred Swiss-Webster (SW) mice as polyuric, polydipsic, glucosuric, and hyperglycemic. Prevalence of glucosuria in the SW colony reached 60% (n=70) in males 8 weeks to 6 months of age. Despite severe obesity in some females, no females were diabetic. Pathologic findings in affected males included cachexia, dilated gastrointestinal tracts with poor muscular tone, pancreatic islet degeneration and atrophy with compensatory metaplasia and/or neogenesis, bacterial pyelonephritis, membranous glomerulopathy, and late-onset hepatic tumors with macrosteatosis, microsteatosis, and hydropic change in aged males. Serum insulin correlated with blood glucose in a nonlinear pattern, suggestive of islet exhaustion. Circulating leptin levels showed a weak inverse correlation with glucose. Diabetic males were bred with obese colony females to produce 20 male and 20 female offspring. Prevalence of diabetes in male offspring was 80% (16/20) with a median age of onset of 18 weeks. By contrast, no diabetic females were identified, despite being significantly more obese than males. Male predominance is likewise a feature of T2D in humans. To our knowledge, this is the first documentation of hepatocellular carcinoma and islet metaplasia and/or neogenesis in a spontaneous outbred mouse model of T2D. The SW availability and histopathologic features represent a promising new model for the study of T2D.
Subject(s)
Carcinoma, Hepatocellular/epidemiology , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/physiopathology , Obesity/physiopathology , Animals , Blood Glucose/metabolism , Body Weight , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Glucose Tolerance Test , Glycosuria , Immunohistochemistry , Insulin/blood , Kaplan-Meier Estimate , Leptin/blood , Male , Mice , Obesity/blood , Obesity/metabolismABSTRACT
BACKGROUND: Comparative characterization of genome-wide transcriptional changes during infection can help elucidate the mechanisms underlying host susceptibility. In this study, transcriptional profiling of the mouse colon was carried out in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N and resistant outbred Swiss Webster mice. Gene expression in the distal colon was determined prior to infection, and at four and nine days post-inoculation using a whole mouse genome Affymetrix array. RESULTS: Computational analysis identified 462 probe sets more than 2-fold differentially expressed between uninoculated resistant and susceptible mice. In response to C. rodentium infection, 5,123 probe sets were differentially expressed in one or both lines of mice. Microarray data were validated by quantitative real-time RT-PCR for 35 selected genes and were found to have a 94% concordance rate. Transcripts represented by 1,547 probe sets were differentially expressed between susceptible and resistant mice regardless of infection status, a host effect. Genes associated with transport were over-represented to a greater extent than even immune response-related genes. Electrolyte analysis revealed reduction in serum levels of chloride and sodium in susceptible animals. CONCLUSION: The results support the hypothesis that mortality in C. rodentium-infected susceptible mice is associated with impaired intestinal ion transport and development of fatal fluid loss and dehydration. These studies contribute to our understanding of the pathogenesis of C. rodentium and suggest novel strategies for the prevention and treatment of diarrhea associated with intestinal bacterial infections.
Subject(s)
Colitis/genetics , Diarrhea/genetics , Dysentery/genetics , Animals , Antiporters/genetics , Carbonic Anhydrase IV/genetics , Citrobacter rodentium , Colitis/microbiology , Colitis/mortality , Diarrhea/microbiology , Diarrhea/mortality , Disease Models, Animal , Dysentery/microbiology , Dysentery/mortality , Electrolytes/blood , Gene Expression Profiling , Genetic Predisposition to Disease , Genomics , Intestines/physiopathology , Ion Transport/genetics , Mice , Mice, Inbred Strains , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Sulfate TransportersABSTRACT
The American bullfrog (Rana catesbeiana) is an aquatic, carnivorous member of the family Ranidae that is used extensively in physiology education programs and in various physiology, toxicology, sensorineural, and genetics research. Eleven bullfrogs purchased from a vendor distributing wild-caught frogs for use in a physiology research protocol were emaciated but otherwise showed no apparent clinical signs of illness. Necropsies performed on selected emaciated frogs indicated heavy infestation with multiple species of endoparasites. Identified helminths included Gorgodera amplicava, Haematolechus breviplexus, Clinostomum spp, Contracaecum spp, Cosmocercoides dukae, and Eustrongyloides spp. Grossly, parasitized bullfrogs showed encysted trematode larvae within skeletal muscle, nematode impaction of the intestinal tract, and lack of coelemic fat stores. Histopathologic lesions were restricted primarily to the gastrointestinal tract and consisted of parasitic granulomas associated with Contracaecum spp. The parasitic lesions may have been associated with the poor body condition of the bullfrogs. Food crickets maintained in-house were negative for parasite larvae or ova. Heavy parasitism of wild-caught bullfrogs may confound research protocols and markedly impair animal health. We encourage researchers to purchase laboratory-bred and -reared bullfrogs and to routinely monitor the parasite status of colony frogs.
Subject(s)
Helminthiasis, Animal/pathology , Helminths/isolation & purification , Nematode Infections/veterinary , Rana catesbeiana/parasitology , Trematode Infections/veterinary , Animals , Female , Gastrointestinal Tract/parasitology , Gastrointestinal Tract/pathology , Granuloma/parasitology , Granuloma/pathology , Helminthiasis, Animal/parasitology , Male , Nematode Infections/parasitology , Nematode Infections/pathology , Trematode Infections/parasitology , Trematode Infections/pathologyABSTRACT
Elevated levels of prostaglandin E(2) (PGE(2)) are often found in colorectal cancers. Thus, nonsteroidal anti-inflammatory drugs, including selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, their long-term use is restricted by the occurrence of adverse events believed to be associated with a global reduction in prostaglandin production. In the present study, we evaluated the chemopreventive efficacy of targeting the terminal synthase microsomal PGE(2) synthase 1 (mPGES-1), which is responsible for generating PGE(2), in two murine models of intestinal cancer. We report for the first time that genetic deletion of mPGES-1 in Apc-mutant mice results in marked and persistent suppression of intestinal cancer growth by 66%, whereas suppression of large adenomas (>3 mm) was almost 95%. This effect occurred despite loss of Apc heterozygosity and beta-catenin activation. However, we found that mPGES-1 deficiency was associated with a disorganized vascular pattern within primary adenomas as determined by CD31 immunostaining. We also examined the effect of mPGES-1 deletion on carcinogen-induced colon cancer. The absence of mPGES-1 reduced the size and number of preneoplastic aberrant crypt foci (ACF). Importantly, mPGES-1 deletion also blocked the nuclear accumulation of beta-catenin in ACF, confirming that beta-catenin is a critical target of PGE(2) procarcinogenic signaling in the colon. Our data show the feasibility of targeting mPGES-1 for cancer chemoprevention with the potential for improved tolerability over traditional nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors.
Subject(s)
Adenoma/genetics , Gene Deletion , Intestinal Neoplasms/genetics , Intramolecular Oxidoreductases/genetics , Animals , Cell Proliferation , Dinoprostone/metabolism , Disease Progression , Female , Homozygote , Intestinal Neoplasms/blood supply , Intestinal Polyps/genetics , Intestinal Polyps/metabolism , Intestinal Polyps/pathology , Intestine, Small/blood supply , Intestine, Small/metabolism , Intramolecular Oxidoreductases/physiology , Isoenzymes/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Precancerous Conditions/genetics , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Prostaglandin-E Synthases , Protein Transport , beta Catenin/metabolismABSTRACT
Sentinel mouse seroconversion to infectious agents is critical for laboratory animal facility disease monitoring. We report spontaneous emergence of non-sex-linked agammaglobulinemia with B-cell deficiency and cutaneous Staphylococcus aureus granulomatosis (botryomycosis) in a cohort of related Swiss Webster sentinel mice. Our experience reinforces the importance of immunocompetency validation in surveillance programs.
Subject(s)
Agammaglobulinemia/complications , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/isolation & purification , Animals , B-Lymphocytes/immunology , Mice , Sentinel Surveillance , Skin/microbiology , Skin/pathologyABSTRACT
Chronic inflammation of mucosal surfaces renders them increasingly susceptible to epithelial cancers both in humans and mice. We have previously shown that anti-inflammatory CD4(+)CD45RB(lo)CD25(+) regulatory (Treg or T(R)) lymphocytes down-regulate inflammation and block development of bacteria-triggered colitis and colorectal cancer (CRC) in 129/SvEv Rag2-/- mice. Interestingly, T(R) cells collected from Interleukin (IL)-10-deficient cell donors not only failed to suppress carcinogenesis but instead promoted invasive mucinous colonic carcinoma with a strong gender bias expressing in male mice. We found we show that peritoneal invasion in this model is dependent on pleiotropic cytokine IL-6. Mucinous carcinoma arose rapidly and consistently after treatment with IL10-/- T(R) cells, which were found to express Foxp3+ and localize throughout tumor tissue. Carcinogenesis was rapidly reversible with transfer of wild type IL10-competent T(R) cells. Likewise, treatment with IL10-Ig fusion protein was sufficient to revert the lesions histologically, and restore inflammatory cytokine and oncogene expression to base line levels. These studies indicate an essential role for IL 6 in this CRC phenotype. Furthermore, immune-competent T(R) cells were important not only for preventing pathology but also for constructive remodeling of bowel following tumorigenic microbial insults. These data provide insights into etiopathogenesis of inflammation-associated epithelial invasion and maintenance of epithelial homeostasis.
Subject(s)
Cell Transformation, Neoplastic/immunology , Colonic Neoplasms/immunology , Helicobacter Infections/microbiology , Interleukin-6/immunology , Animals , CD4 Antigens/immunology , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Colonic Neoplasms/microbiology , Colonic Neoplasms/pathology , Helicobacter Infections/immunology , Helicobacter Infections/metabolism , Helicobacter hepaticus , Interleukin-10/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Male , Mice , Mice, Knockout , Neoplasm Invasiveness , T-Lymphocytes, Regulatory/immunologyABSTRACT
Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia. The disease is characterized by severe but temporary epithelial hyperplasia with limited inflammation in the descending colon of adult mice on a variety of genetic backgrounds. The natural history of infection with this murine pathogen has been characterized in outbred Swiss Webster (SW) mice but not in the cognate inbred FVB strain. In contrast to subclinical infection in SW mice, 12-week-old FVB mice developed overt disease with significant weight loss and mortality beginning by 9 days postinoculation (dpi). By 21 dpi, more than 75% of infected FVB mice died or had to be euthanized, whereas no mortality developed in SW mice. Mortality in FVB mice was fully prevented by fluid therapy. Fecal shedding of bacteria was similar in both groups through 9 dpi; however, a slight but significant delay in bacterial clearance was observed in FVB mice by 12 to 18 dpi. SW mice developed hyperplasia with minimal inflammation in the descending colon. FVB mice developed epithelial cell hyperproliferation, severe inflammation with erosions and ulcers, and epithelial atypia by 6 dpi in the descending colon. In the majority of surviving FVB mice, colonic lesions, including epithelial atypia, were reversible, although a small percentage (5 to 7%) exhibited chronic colitis through 7 months postinoculation. The existence of susceptible and resistant lines of mice with similar genetic backgrounds will facilitate the identification of host factors responsible for the outcome of infection and may lead to the development of novel strategies for preventing and treating infectious colitis.
Subject(s)
Citrobacter rodentium/pathogenicity , Colitis/mortality , Colitis/pathology , Disease Models, Animal , Animals , Animals, Inbred Strains , Animals, Outbred Strains , Citrobacter rodentium/genetics , Citrobacter rodentium/isolation & purification , Colitis/microbiology , Colon/cytology , Colon/microbiology , Colon/pathology , Colony Count, Microbial , Epithelial Cells/microbiology , Epithelial Cells/pathology , Female , Humans , Hyperplasia/microbiology , Hyperplasia/pathology , Inflammation , Male , Mice , Mice, Mutant Strains , Species Specificity , Specific Pathogen-Free OrganismsABSTRACT
Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2-/- cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2-/- lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.
Subject(s)
Gastric Mucosa/immunology , Gastrointestinal Diseases/immunology , Inflammation/immunology , Lymphocytes/immunology , Macrophages/immunology , Mucins/immunology , Muscle Proteins/immunology , Peptides/immunology , Animals , Cytokines/immunology , Enzyme-Linked Immunosorbent Assay , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gastrointestinal Diseases/microbiology , Gastrointestinal Diseases/prevention & control , Gene Expression , Gene Expression Profiling , Helicobacter Infections/immunology , Helicobacter Infections/metabolism , Helicobacter Infections/pathology , Helicobacter felis/immunology , Inflammation/microbiology , Inflammation/pathology , Intestinal Mucosa/metabolism , Intestines/immunology , Intestines/pathology , Lymphocytes/microbiology , Macrophages/microbiology , Mice , Mice, Knockout , Mucins/genetics , Mucins/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Peptides/genetics , Peptides/metabolism , Receptors, Interleukin-1/immunology , Reverse Transcriptase Polymerase Chain Reaction , Stomach/immunology , Stomach/pathology , Trefoil Factor-2ABSTRACT
Defects within the innate immune system sensitize NF-kappaB-deficient (p50(-/-); p65(+/-)) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-kappaB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2(-/-) (RAG) and p50(-/-); p65(+/-); RAG-2(-/-) (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50(-/-); RAG-2(-/-) (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-kappaB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.
Subject(s)
Colitis/immunology , Colitis/prevention & control , Helicobacter Infections/immunology , Helicobacter Infections/prevention & control , Helicobacter hepaticus/immunology , Interleukin-10/physiology , NF-kappa B p50 Subunit/physiology , Animals , Colitis/genetics , Colitis/microbiology , Colon/immunology , Colon/microbiology , Helicobacter Infections/genetics , Helicobacter hepaticus/growth & development , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Inflammation Mediators/immunology , Inflammation Mediators/physiology , Inflammation Mediators/therapeutic use , Interleukin-10/deficiency , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12 Subunit p40/biosynthesis , Interleukin-12 Subunit p40/genetics , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Knockout , NF-kappa B p50 Subunit/deficiency , NF-kappa B p50 Subunit/genetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/therapeutic useABSTRACT
Microaerobic bacteria were isolated from a baboon with pancreatic islet amyloidosis and hepatitis. Phenotypic and molecular analyses identified two distinct helicobacters. Analyses of 16S rRNA demonstrated "Helicobacter macacae" in the ileum and liver, and Helicobacter cinaedi in the colon. To the best of the authors' knowledge, this is the first report describing the isolation of enterohepatic Helicobacter species from a baboon.