Cannabinoid control of gingival immune activation in chronically SIV-infected rhesus macaques involves modulation of the indoleamine-2,3-dioxygenase-1 pathway and salivary microbiome.

BACKGROUND: HIV/SIV-associated periodontal disease (gingivitis/periodontitis) (PD) represents a major comorbidity affecting people living with HIV (PLWH) on combination anti-retroviral therapy (cART). PD is characterized by chronic inflammation and dysbiosis. Nevertheless, the molecular mechanisms and use of feasible therapeutic strategies to reduce/reverse inflammation and dysbiosis remain understudied and unaddressed. METHODS: Employing a systems biology approach, we report molecular, metabolome and microbiome changes underlying PD and its modulation by phytocannabinoids [delta-9-tetrahydrocannabinol (Δ9-THC)] in uninfected and SIV-infected rhesus macaques (RMs) untreated (VEH-untreated/SIV) or treated with vehicle (VEH/SIV) or Δ9-THC (THC/SIV). FINDINGS: VEH- untreated/SIV but not THC/SIV RMs showed significant enrichment of genes linked to anti-viral defense, interferon-ß, NFκB, RIG-1, and JAK-STAT signaling. We focused on the anti-microbial DUOX1 and immune activation marker IDO1 that were reciprocally regulated in the gingiva of VEH-untreated/SIV RMs. Both proteins localized to the gingival epithelium and CD163+ macrophages, and showed differential expression in the gingiva of THC/SIV and VEH/SIV RMs. Additionally, inflammation-associated miR-21, miR-142-3p, miR-223, and miR-125a-5p showed significantly higher expression in the gingiva of VEH/SIV RMs. In human primary gingival epithelial cells, miR-125a-5p post-transcriptionally downregulated DUOX1 and THC inhibited IDO1 protein expression through a cannabinoid receptor-2 mediated mechanism. Interestingly, THC/SIV RMs showed relatively reduced plasma levels of kynurenine, kynurenate, and the neurotoxic quinolinate compared to VEH/SIV RMs at 5 months post SIV infection (MPI). Most importantly, THC blocked HIV/SIV-induced depletion of Firmicutes and Bacteroidetes, and reduced Gammaproteobacteria abundance in saliva. Reduced IDO1 protein expression was associated with significantly (p<0.05) higher abundance of Prevotella, Lactobacillus (L. salivarius, L. buchneri, L. fermentum, L. paracasei, L. rhamnosus, L. johnsonii) and Bifidobacteria and reduced abundance of the pathogenic Porphyromonas cangingivalis and Porphyromonas macacae at 5MPI. INTERPRETATION: The data provides deeper insights into the molecular mechanisms underlying HIV/SIV-induced PD and more importantly, the anti-inflammatory and anti-dysbiotic properties of THC in the oral cavity. Overall, these translational findings suggest that phytocannabinoids may help reduce gingival/systemic inflammation, salivary dysbiosis and potentially metabolic disease/syndrome in PLWH on cART and those with no access to cART or do not suppress the virus under cART. FUNDING: Research reported in this publication was supported by the National Institutes of Health Award Numbers R01DA052845 (MM and SNB), R01DA050169 (MM and CO), R01DA042524 and R56DE026930 (MM), and P51OD011104 and P51OD011133. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Dietary Gluten and Neurodegeneration: A Case for Preclinical Studies.

Although celiac disease (CD) is an autoimmune disease that primarily involves the intestinal tract, mounting evidence suggests that a sizeable number of patients exhibit neurological deficits. About 40% of the celiac patients with neurological manifestations have circulating antibodies against neural tissue transglutaminase-6 (tTG6). While early diagnosis and strict adherence to a gluten-free diet (GFD) have been recommended to prevent neurological dysfunction, better therapeutic strategies are needed to improve the overall quality of life. Dysregulation of the microbiota-gut-brain axis, presence of anti-tTG6 antibodies, and epigenetic mechanisms have been implicated in the pathogenesis. It is also possible that circulating or gut-derived extracellular structures and including biomolecular condensates and extracellular vesicles contribute to disease pathogenesis. There are several avenues for shaping the dysregulated gut homeostasis in individuals with CD, non-celiac gluten sensitivity (NCGS) and/or neurodegeneration. In addition to GFD and probiotics, nutraceuticals, such as phyto and synthetic cannabinoids, represent a new approach that could shape the host microbiome towards better prognostic outcomes. Finally, we provide a data-driven rationale for potential future pre-clinical research involving non-human primates (NHPs) to investigate the effect of nutraceuticals, such as phyto and synthetic cannabinoids, either alone or in combination with GFD to prevent/mitigate dietary gluten-induced neurodegeneration.

Long Term Delta-9-tetrahydrocannabinol Administration Inhibits Proinflammatory Responses in Minor Salivary Glands of Chronically Simian Immunodeficieny Virus Infected Rhesus Macaques.

HIV/SIV-associated oral mucosal disease/dysfunction (HAOMD) (gingivitis/periodontitis/salivary adenitis) represents a major comorbidity affecting HIV patients on anti-retroviral therapy. Using a systems biology approach, we investigated molecular changes (mRNA/microRNA) underlying HAOMD and its modulation by phytocannabinoids (delta-9-tetrahydrocannabinol (∆9-THC)) in uninfected (n = 5) and SIV-infected rhesus macaques untreated (VEH-untreated/SIV; n = 7) or treated with vehicle (VEH/SIV; n = 3) or ∆9-THC (THC/SIV; n = 3). Relative to controls, fewer mRNAs were upregulated in THC/SIV compared to VEH-untreated/SIV macaques. Gene enrichment analysis showed differential enrichment of biological functions involved in anti-viral defense, Type-I interferon, Toll-like receptor, RIG-1 and IL1R signaling in VEH-untreated/SIV macaques. We focused on the anti-ER-stress anterior gradient-2 (AGR2), epithelial barrier protecting and anti-dysbiotic WAP Four-Disulfide Core Domain-2 (WFDC2) and glucocorticoid-induced anti-inflammatory TSC22D3 (TSC22-domain family member-3) that were significantly downregulated in oropharyngeal mucosa (OPM) of VEH-untreated/SIV macaques. All three proteins localized to minor salivary gland acini and secretory ducts and showed enhanced and reduced expression in OPM of THC/SIV and VEH/SIV macaques, respectively. Additionally, inflammation associated miR-21, miR-142-3p and miR-29b showed significantly higher expression in OPM of VEH-untreated/SIV macaques. TSC22D3 was validated as a target of miR-29b. These preliminary translational findings suggest that phytocannabinoids may safely and effectively reduce oral inflammatory responses in HIV/SIV and other (autoimmune) diseases.

Rotavirus Reassortant-Induced Murine Model of Liver Fibrosis Parallels Human Biliary Atresia.

BACKGROUND AND AIMS: Biliary atresia (BA) is a devastating neonatal cholangiopathy that progresses to fibrosis and end-stage liver disease by 2 years of age. Portoenterostomy may reestablish biliary drainage, but, despite drainage, virtually all afflicted patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. APPROACH AND RESULTS: In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling human BA and has been used to study mechanistic aspects of the disease. Unfortunately, nearly all RRV-infected pups succumb by day of life 14. Thus, in this study we generated an RRV-TUCH rotavirus reassortant (designated as TR(VP2,VP4) ) that when injected into newborn mice causes an obstructive jaundice phenotype with lower mortality rates. Of the mice that survived, 63% developed Ishak stage 3-5 fibrosis with histopathological signs of inflammation/fibrosis and bile duct obstruction. CONCLUSIONS: This model of rotavirus-induced neonatal fibrosis will provide an opportunity to study disease pathogenesis and has potential to be used in preclinical studies with an objective to identify therapeutic targets that may alter the course of BA.

Non-Human Primate Models of Enteric Viral Infections.

There is an important role non-human primates (NHP) play in biomedical research. Phylogenetic proximity of any of the NHP species to Homo sapiens assures that much better translatability of research outcomes from model studies involving human diseases can be achieved than from those generated with other pre-clinical systems. Our group and others used during past two decades NHPs in research directed towards viral and autoimmune disorders of the gastrointestinal tract. This review summarizes progress made in the area of enteric viral infections including its applicability to human disease.

Beneficial Effects of Human Anti-Interleukin-15 Antibody in Gluten-Sensitive Rhesus Macaques with Celiac Disease.

Overexpression of interleukin-15 (IL-15) is linked with immunopathology of several autoimmune disorders including celiac disease. Here, we utilized an anti-human IL-15 antibody 04H04 (anti-IL-15) to reverse immunopathogenesis of celiac disease. Anti-IL-15 was administered to six gluten-sensitive rhesus macaques with celiac disease characteristics including gluten-sensitive enteropathy (GSE), and the following celiac-related metrics were evaluated: morphology (villous height/crypt depth ratio) of small intestine, counts of intestinal intraepithelial lymphocytes, IFN-γ-producing CD8+ and CD4+ T cells, plasma levels of anti-gliadin and anti-intestinal tissue transglutaminase IgG antibodies, as well as peripheral effector memory (CD3+CD28-CD95+) T cells. Anti-IL-15 treatment reversed the clinically relevant disease endpoints, intraepithelial lymphocyte counts, and villous height/crypt depth ratios within jejunal biopsies to normal levels (P < 0.001). Additionally, intestinal CD8+ and CD4+ T cell IFN-γ production was reduced (P < 0.05). Extra-intestinally, anti-IL-15 treatment reduced peripheral NK cell counts (P < 0.001), but otherwise, non-NK peripheral lymphocytes including effector memory T cells and serum blood chemistry were unaffected. Overall, providing the beneficial disease-modulatory and immunomodulatory effects observed, anti-IL-15 treatment might be considered as a novel therapy to normalize intestinal lymphocyte function in celiac disease patients with GSE.

The SRL peptide of rhesus rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia.

Biliary atresia (BA) is a neonatal obstructive cholangiopathy that progresses to end-stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that the RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Using rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains that cause obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4-derived peptide (TRTRVSRLY) significantly reduced the ability of RRV to bind and infect cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains that do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by small interfering RNAs reduced RRV's ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. CONCLUSION: The tripeptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70, defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response to this interaction to understand how it contributes to the pathogenesis of BA. (Hepatology 2017;65:1278-1292).

Dietary Gluten-Induced Gut Dysbiosis Is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease.

The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS) captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD), gluten-free diet (GFD), barley gluten-derived diet (BOMI) and reduced gluten barley-derived diet (RGB). It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p < 0.05) following the consumption of dietary gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae) were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity) of gut microbiome in GS macaques started to change (p = 0.011) towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b) were upregulated (p < 0.05) in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911), Prevotella stercorea (NR_041364) and Streptococcus luteciae (AJ297218) that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes in GS macaques will approach those of healthy individuals. Further studies are needed to elucidate the regulatory pathways of inflammatory miRNAs in intestinal mucosa of GS macaques and to correlate their expression with gut dysbiosis.

Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques.

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement-but not remission-of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm-by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-γ and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea-all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

The effects of reduced gluten barley diet on humoral and cell-mediated systemic immune responses of gluten-sensitive rhesus macaques.

Celiac disease (CD) affects approximately 1% of the general population while an estimated additional 6% suffers from a recently characterized, rapidly emerging, similar disease, referred to as non-celiac gluten sensitivity (NCGS). The only effective treatment of CD and NCGS requires removal of gluten sources from the diet. Since required adherence to a gluten-free diet (GFD) is difficult to accomplish, efforts to develop alternative treatments have been intensifying in recent years. In this study, the non-human primate model of CD/NCGS, e.g., gluten-sensitive rhesus macaque, was utilized with the objective to evaluate the treatment potential of reduced gluten cereals using a reduced gluten (RG; 1% of normal gluten) barley mutant as a model. Conventional and RG barleys were used for the formulation of experimental chows and fed to gluten-sensitive (GS) and control macaques to determine if RG barley causes a remission of dietary gluten-induced clinical and immune responses in GS macaques. The impacts of the RG barley diet were compared with the impacts of the conventional barley-containing chow and the GFD. Although remission of the anti-gliadin antibody (AGA) serum responses and an improvement of clinical diarrhea were noted after switching the conventional to the RG barley diet, production of inflammatory cytokines, e.g., interferon-gamma (IFN-γ), tumor necrosis factor (TNF) and interleukin-8 (IL-8) by peripheral CD4+ T helper lymphocytes, persisted during the RG chow treatment and were partially abolished only upon re-administration of the GFD. It was concluded that the RG barley diet might be used for the partial improvement of gluten-induced disease but its therapeutic value still requires upgrading-by co-administration of additional treatments.

Role of histo-blood group antigens in primate enteric calicivirus infections.

Human noroviruses (NoV) are associated with large proportion of non-bacterial diarrhea outbreaks together with > 50% of food-associated diarrheas. The function of histo-blood group antigens (HBGAs) in pathogenesis of virus infection was implicated. Until recently however, due to lack of a robust animal and in vitro models of human NoV infection, only the partial knowledge concerning the virus pathogenesis (receptor, co-receptor and target cell) and absence of viable vaccine candidates were the frequently referenced attributes of this acute diarrheal illness. Recently, a novel group of enteric caliciviruses (CV) of rhesus macaque host origin was discovered and described. The new genus within the family Caliciviridae was identified: Rhesus Enteric CV, i.e., "Recovirus" (ReCV). ReCVs are genetically and biologically close relatives of human NoVs, exhibit similar genetic and biological features and are capable of being propagated in cell culture. ReCVs cause symptomatic disease (diarrhea and fever) in experimentally inoculated macaques. Formulation and evaluation of efficient NoV vaccine might take several years. As suggested by recent studies, inhibition of HBGAs or HBGA-based antivirals could meanwhile be exploited as vaccine alternatives. The purpose of this minireview is to provide the guidance in respect to newly available primate model of enteric CV infection and its similarities with human NoV in utilizing the HBGAs as potential virus co-receptors to indirectly address the unresolved questions of NoV pathogenesis and immunity.

Transmissible gastroenteritis virus: identification of M protein-binding peptide ligands with antiviral and diagnostic potential.

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p<0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment.

Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease.

Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin-) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p<0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.

Celiac and Non-Celiac Forms of Gluten Sensitivity: Shifting Paradigms of an Old Disease.

Gluten sensitivity is one of the prominent features of celiac disease (CD) which is an autoimmune disorder characterized by damaged lining of the small intestine. CD was known already to ancient Greeks as κοιλιακός (keeleeakoss) i.e. disease of the abdominal cavity hence celiac. Focus of this Commentary article is on rather complex definition of CD and its emerging new forms the example of which is non-celiac gluten sensitivity. It is becoming evident that to formulate more effective treatments, these associations and newly identified disease entities deserve attention from both academic and clinical communities.

High incidence of rhesus enteric calicivirus infections and diarrhea in captive juvenile macaques: a likely association.

BACKGROUND: The rhesus enteric caliciviruses (ReCVs) were recently described. METHODS: Prevalence of ReCV antibodies was tested in six species of captive non-human primates. RESULTS: High ReCV seroprevalence was revealed in rhesus and cynomolgus macaques. CONCLUSIONS: High rates of ReCV seroprevalence and diarrhea in juvenile macaques suggest that ReCVs may play a role in morbidity.

Rotavirus infection of human cholangiocytes parallels the murine model of biliary atresia.

INTRODUCTION: Biliary atresia (BA) is the leading indication for liver transplantation in the pediatric population. The murine model of BA supports a viral etiology, because infection of neonatal mice with rhesus rotavirus (RRV) results in biliary obstruction. Viral infection targets the biliary epithelium and development of the model is viral strain dependent. No study has yet determined whether human cholangiocytes are also susceptible to rotaviral infection. We established an in vitro human model using an immortalized human cholangiocyte cell line and primary human cholangiocytes obtained from explanted livers to determine human cholangiocyte susceptibility to rotavirus infection. METHODS: Replication and binding assays were performed on immortalized mouse (mCL) and human (H69) cells using six different strains of rotavirus. Primary human cholangiocytes were isolated from cadaveric livers, characterized in culture, and infected with RRV, which causes BA in mice, and another simian strain, TUCH, which does not cause BA in mice. RESULTS: Immortalized mouse and human cholangiocytes demonstrated similar patterns of infectivity and binding with different strains of rotavirus. Both cell lines produced a significantly higher viral yield with RRV infection than with the other strains tested. In primary human cholangiocytes, which maintained their epithelial characteristics, as demonstrated by cytokeratin staining, RRV replicated to a yield 1000-fold higher than TUCH. CONCLUSIONS: Both immortalized and primary human cholangiocytes are susceptible to RRV infection in a fashion similar to murine cholangiocytes. These novel findings suggest rotavirus infection could have a potential role in the pathogenesis of human BA.

Experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses.

BACKGROUND: Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity.

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice.

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.

Improved xenobiotic metabolism and reduced susceptibility to cancer in gluten-sensitive macaques upon introduction of a gluten-free diet.

BACKGROUND: A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta). METHODOLOGY: Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes. PRINCIPAL FINDINGS: When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories--cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function--this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes. CONCLUSIONS/SIGNIFICANCE: A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research.