Cryptosporidium Parvum-Induced Inflammatory Bowel Disease of TCR-β- x TCR-δ-Deficient Mice

Experimental inoculation of neonatal immunocompetent strains of mice with Cryptosporidium parvum results in a transient, noninflammatory enteric infection. In the present study, we show that inoculation of mice deficient in a 3 and y8 T cells (TCR-3- X TCR-8-deficient mice) with C. parvum results in persistent infection and severe inflammatory bowel disease- like lesions. The most severe lesions in these mice were in the cecum with similar yet less severe lesions in the ileum and proximal colon. The most notable aspect of the histopathology was glandular hyperplasia with abscess formation, extensive fibrosis of the lamina propria with infiltrates of predominately polymorphonuclear cells and macrophages, and a few small aggregates of B cells. Persistently infected mice also developed extensive hepatic periportal fibrosis in association with C. parvum colonization of bile ducts. Lesions observed in TCR- 3- X TCR-8-deficient mice were markedly different than previously described lesions detected in C. parvum-infected TCR-o-deficient mice. Cryptosporidium parvum-infected TCR-o-deficient mice have extensive infiltrations of B cells, whereas TCR-P3- X TCR-8-deficient mice had only a few small aggregates of B cells. These findings indicate that although y8 T cells are not necessary for induction of intestinal inflammation in C. parvum-infected o(4 T- cell-deficient mice, their presence does alter the morphology of the ensuing lesion

Transport of Artificial Virus-like Nanocarriers (AVN) through intestinal monolayer via Microfold cells

Compared with subcutaneous or intramuscular routes for vaccination, vaccine delivery via gastrointestinal mucosa has tremendous potential as it is easy to administer and pain free. Robust immune responses can be triggered successfully once vaccine carried antigen reaches the mucosal associated lymphoid sites (e.g., Peyer’s patches). However, the absence of an efficient delivery method has always been an issue for successful oral vaccine development. In our study, inspired by mammalian orthoreovirus (MRV) transport into gut mucosal lymphoid tissue via Microfold cells (M cells), artificial virus-like nanocarriers (AVN), consisting of gold nanocages functionalized with the 1 protein from mammalian reovirus (MRV), were tested as an effective oral vaccine delivery vehicle targeting M cells. AVN was shown to have a significantly higher transport compared to other experimental groups across mouse organoid monolayers containing M cells. These findings suggest that AVN has the potential to be an M cell-specific oral vaccine/drug delivery vehicle

Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice

Background: Helicobacter bilis infection of C3H/HeN mice harboring the altered Schaedler flora (ASF) triggers progressive immune responsiveness and the development of colitis. We sought to investigate temporal alterations in community structure of a defined (ASF-colonized) microbiota in normal and inflamed murine intestines and to correlate microbiota changes to histopathologic lesions. Methods: The colonic mucosal microbiota of healthy mice and ASF mice colonized with H. bilis for 3, 6, or 12 weeks were investigated by fluorescence in situ hybridization targeting the 16S ribosomal RNA genes of total bacteria, group-specific organisms, and individual ASF bacterial species. Microbial profiling of ASF and H. bilis abundance was performed on cecal contents. Results: Helicobacter bilis–colonized mice developed colitis associated with temporal changes in composition and spatial distribution of the mucosal microbiota. The number of total bacteria, ASF519, and helicobacter-positive bacteria were increased (P , 0.05), whereas ASF360/361-positive bacteria were decreased (P , 0.05) versus controls. Adherent biofilms in colitic mice were most often (P , 0.05) composed of total bacteria, ASF457, and H. bilis. Total numbers of ASF519 and H. bilis bacteria were positively correlated (P ¼ 0.03, r ¼ 0.39 and P , 0.0001, r ¼ 0.73), and total numbers of ASF360/361 bacteria were negatively correlated (P ¼ 0.003, r ¼ 20.53) to histopathologic score. Differences in cecal abundance of ASF members were not observed. Conclusions: Altered community structure with murine colitis is characterized by distinct ASF bacteria that interact with the colonic mucosa, by formation of an isolating interlaced layer, by attachment, or by invasion, and this interaction is differentially expressed over time

Pentablock copolymer micelle nanoadjuvants enhance cytosolic delivery of antigen and improve vaccine efficacy while inducing low inflammation

As the focus has shifted from traditional killed or live, attenuated vaccines towards subunit vaccines, improvements in vaccine safety have been confronted with low immunogenicity of protein antigens. This issue has been addressed by synthesizing and designing a wide variety of antigen carriers and adjuvants, such as Toll-like receptor agonists (e.g., MPLA, CpG). Studies have focused on optimizing adjuvants for improved cellular trafficking, cytosolic availability, and improved antigen presentation. In this work, we describe the design of novel amphiphilic pentablock copolymer (PBC) adjuvants that exhibit high biocompatibility and reversible pH- and temperature-sensitive micelle formation. We demonstrate improved humoral immunity in mice in response to single dose immunization with PBC micelle adjuvants compared to soluble antigen alone. With the motive of exploring the mechanism of action of these PBC micelles, we studied intracellular trafficking of these PBC micelles with a model antigen and demonstrated that the PBC micelles associate with the antigen and enhance its cytosolic delivery to antigen presenting cells. We posit that these PBC micelles operate via immune-enhancing mechanisms that are different from that of traditional Toll-like receptor activating adjuvants. The metabolic profile of antigen presenting cells stimulated with traditional adjuvants and the PBC micelles also suggests distinct mechanisms of action. A key finding from this study is the low production of nitric oxide and reactive oxygen species by antigen presenting cells when stimulated by PBC micelle adjuvants in sharp contrast to TLR adjuvants. Together, these studies provide a basis for rationally developing novel vaccine adjuvants that are safe, that induce low inflammation, and that can efficiently deliver antigen to the cytosol

Vitamin C and B3 as New Biomaterials to Alter Intestinal Stem Cells

Vitamin C (ascorbic acid) and vitamin B3 (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact towards homeostasis and epigenetic changes.1 Vitamin B3 aids in maintaining healthy intestinal homeostasis and reducing gut inflammation by participating in the rapamycin signaling pathway.2 In this study, vitamin C and vitamin B3 (600 and 1200 μg/ml) have been explored as potential new biomaterials to study their effects on four types of intestinal stem cells which are isolated from mice bearing different microbiota. We observed that C3H ASF and 129 ASF IL‐10 are more sensitive towards 600 μg/ml vitamin B3 and 1200 μg/ml vitamin C. The lowest growth rate and viability for all types of organoids was with 1200 μg/ml vitamin C. From qPCR analysis (quantitative Polymerase Chain Reaction analysis), MUC2 was upregulated for 129 ASF and C3H Conv when exposed to 600 μg/ml and 1200 μg/ml vitamin C. It suggests that large amounts of glycoprotein may be produced after adding high concentrations of vitamin C. Since inflammatory bowel disease has low level of MUC2, this finding may be helpful in restoring mucosal health by upregulating the MUC2 gene while altering patient\u27s microbiota.3 These results are expected to have a positive translational impact because this bottom‐up strategy would be instrumental in developing Vitamin C and B3 based orally available therapeutic strategies and formula for advancing the fields of gastrointestinal regenerative medicine

Temporal Dynamics of Chronic Inflammation on the Cecal Microbiota in IL-10-/- Mice

The intestinal microbiota is a critical component of mucosal health as evidenced by the fact that alterations in the taxonomic composition of the gastrointestinal microbiota are associated with inflammatory bowel diseases. To better understand how the progression of inflammation impacts the composition of the gastrointestinal microbiota, we used culture independent taxonomic profiling to identify temporal changes in the cecal microbiota of C3Bir IL-10-/- mice concomitantly with the onset and progression of colitis. This analysis revealed that IL-10-/- mice displayed a biphasic progression in disease severity, as evidenced by histopathological scores and cytokine production. Beginning at 4 weeks of age, pro-inflammatory cytokines including TNF-a, IFN-g, IL-6, G- CSF, and IL-1a as well as chemokines including RANTES and MIP-1a were elevated in the serum of IL-10-/- mice. By 19 weeks of age, the mice developed clinical signs of disease as evidenced by weight loss, which was accompanied by a significant increase in serum levels of KC and IL-17. While the overall diversity of the microbiota of both wild type and IL-10-/- were similar in young mice, the latter failed to increase in complexity as the mice matured and experienced changes in abundance of specific bacterial taxa that are associated with inflammatory bowel disease in humans. Collectively, these results reveal that there is a critical time in young mice between four to six weeks of age when inflammation and the associated immune responses adversely affect maturation of the microbiota

Amphiphilic polyanhydride-based recombinant MUC4β-nanovaccine activates dendritic cells

Mucin 4 (MUC4) is a high molecular weight glycoprotein that is differentially overexpressed in pancreatic cancer (PC), functionally contributes to disease progression, and correlates with poor survival. Further, due to its aberrant glycosylation and extensive splicing, MUC4 is a potential target for cancer immunotherapy. Our previous studies have demonstrated the utility of amphiphilic polyanhydride nanoparticles as a useful platform for the development of protein-based prophylactic and therapeutic vaccines. In the present study, we encapsulated purified recombinant human MUC4-beta (MUC4β) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4β-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4β-nanovaccine exhibited significant increase in the surface expressions of MHC I and MHC II and costimulatory molecules (CD80 and CD86), as well as, secretion of pro-inflammatory cytokines (IFN-γ, IL-6, and IL-12) as compared to cells exposed to MUC4β alone or MUC4β mixed with blank nanoparticles (MUC4β+NP). Following immunization, as compared to the other formulations, MUC4β-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4β-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4β-nanovaccine as a novel platform for PC immunotherapy

Polyanhydride Nanoparticles Induce Low Inflammatory Dendritic Cell Activation Resulting in CD8+ T Cell Memory and Delayed Tumor Progression

Introduction: Adjuvants and immunotherapies designed to activate adaptive immunity to eliminate infectious disease and tumors have become an area of interest aimed at providing a safe and effective strategy to prevent or eliminate disease. Existing approaches would benefit from the development of immunization regimens capable of inducing efficacious cell-mediated immunity directed toward CD8+ T cell-specific antigens. This goal is critically dependent upon appropriate activation of antigen-presenting cells (APCs) most notably dendritic cells (DCs). In this regard, polyanhydride particles have been shown to be effectively internalized by APCs and induce activation.Methods: Here, a prophylactic vaccine regimen designed as a single-dose polyanhydride nanovaccine encapsulating antigen is evaluated for the induction of CD8+ T cell memory in a model system where antigen-specific protection is restricted to CD8+ T cells. Bone marrow-derived dendritic cells (BMDCs) are used as an in vitro model system to evaluate the magnitude and phenotype of APC activation. Primary DCs, particularly those with described ability to activate CD8+ T cells, are also evaluated for their in vitro responses to polyanhydride nanoparticles.Results: Herein, polyanhydride nanoparticles are shown to induce potent in vitro upregulation of costimulatory molecules on the cell surface of BMDCs. In contrast to the classically used TLR agonists, nanoparticles did not induce large amounts of pro-inflammatory cytokines, did not induce characteristic metabolic response of DCs, nor produce innate antimicrobial effector molecules, such as nitric oxide (NO). The polyanhydride nanovaccine results in protective CD8+ T cell responses as measured by inhibition of tumor progression and survival.Discussion: Together, these results suggest that the use of a polyanhydride-based nanovaccine can be an effective approach to inducing antigen-specific CD8+ T cell memory by providing antigen delivery and DC activation while avoiding overt inflammatory responses typically associated with traditional adjuvants

Gene expression in intestinal mucosal biopsy specimens obtained from dogs with chronic enteropathy

Objective—To characterize mucosal gene expression in dogs with chronic enteropathy (CE). Animals—18 dogs with CE and 6 healthy control dogs. Procedures—Small intestinal mucosal biopsy specimens were endoscopically obtained from dogs. Disease severity in dogs with CE was determined via inflammatory bowel index scores and histologic grading of biopsy specimens. Total RNA was extracted from biopsy specimens and microchip array analysis (approx 43,000 probe sets) and quantitative reverse transcriptase PCR assays were performed. Results—1,875 genes were differentially expressed between dogs with CE and healthy control dogs; 1,582 (85%) genes were downregulated in dogs with CE, including neurotensin, fatty acid–binding protein 6, fatty acid synthase, aldehyde dehydrogenase 1 family member B1, metallothionein, and claudin 8, whereas few genes were upregulated in dogs with CE, including genes encoding products involved in extracellular matrix degradation (matrix metallopeptidases 1, 3, and 13), inflammation (tumor necrosis factor, interleukin-8, peroxisome proliferator–activated receptor γ, and S100 calcium-binding protein G), iron transport (solute carrier family 40 member 1), and immunity (CD96 and carcinoembryonic antigen–related cell adhesion molecule [CEACAM] 18). Dogs with CE and protein-losing enteropathy had the greatest number of differentially expressed genes. Results of quantitative reverse transcriptase PCR assay for select genes were similar to those for microchip array analysis. Conclusions and Clinical Relevance—Expression of genes encoding products regulating mucosal inflammation was altered in dogs with CE and varied with disease severity. Impact for Human Medicine—Molecular pathogenesis of CE in dogs may be similar to that in humans with inflammatory bowel disease

Gene expression in intestinal mucosal biopsy specimens obtained from dogs with chronic enteropathy

Objective—To characterize mucosal gene expression in dogs with chronic enteropathy (CE). Animals—18 dogs with CE and 6 healthy control dogs. Procedures—Small intestinal mucosal biopsy specimens were endoscopically obtained from dogs. Disease severity in dogs with CE was determined via inflammatory bowel index scores and histologic grading of biopsy specimens. Total RNA was extracted from biopsy specimens and microchip array analysis (approx 43,000 probe sets) and quantitative reverse transcriptase PCR assays were performed. Results—1,875 genes were differentially expressed between dogs with CE and healthy control dogs; 1,582 (85%) genes were downregulated in dogs with CE, including neurotensin, fatty acid–binding protein 6, fatty acid synthase, aldehyde dehydrogenase 1 family member B1, metallothionein, and claudin 8, whereas few genes were upregulated in dogs with CE, including genes encoding products involved in extracellular matrix degradation (matrix metallopeptidases 1, 3, and 13), inflammation (tumor necrosis factor, interleukin-8, peroxisome proliferator–activated receptor γ, and S100 calcium-binding protein G), iron transport (solute carrier family 40 member 1), and immunity (CD96 and carcinoembryonic antigen–related cell adhesion molecule [CEACAM] 18). Dogs with CE and protein-losing enteropathy had the greatest number of differentially expressed genes. Results of quantitative reverse transcriptase PCR assay for select genes were similar to those for microchip array analysis. Conclusions and Clinical Relevance—Expression of genes encoding products regulating mucosal inflammation was altered in dogs with CE and varied with disease severity. Impact for Human Medicine—Molecular pathogenesis of CE in dogs may be similar to that in humans with inflammatory bowel disease