Concerns about the histological assessment in a mouse model of human celiac disease.

Commentary on: Abadie V et al. IL­15, gluten and HLA­DQ8 drive tissue destruction in coeliac disease. Nature. 2020; 578: 600­604

In vitro adhesion, pilus expression, and in vivo amelioration of antibiotic-induced microbiota disturbance by Bifidobacterium spp. strains from fecal donors.

Fecal microbiota transplantation (FMT) is used routinely to treat recurrent Clostridioides difficile infection (rCDI) and investigated as a treatment for numerous conditions associated with gut microbiota alterations. Metagenomic analyses have indicated that recipient colonization by donor bacteria may be associated with favorable clinical outcomes. Bifidobacteria are abundant gut commensals associated with health. We have previously demonstrated that Bifidobacterium strains transferred in FMT can colonize recipients in long term, at least for a year, and recovered such strains by cultivation. This study addressed in vitro adhesion and pilus gene expression of long-term colonizing Bifidobacterium strains from FMT donors as well as in vivo colonization and capability to ameliorate antibiotic-induced microbiota disturbance. RNA-Seq differential gene expression analysis showed that the strongly adherent B. longum strains DY_pv11 and DX_pv23 expressed tight adherence and sortase-dependent pilus genes, respectively. Two B. longum strains, adherent DX_pv23 and poorly adhering DX_pv18, were selected to address in vivo colonization and efficacy to restore antibiotic-disturbed microbiota in C57BL/6 murine model. DX_pv23 colonized mice transiently with a rate comparable to that of the B. animalis BB-12 used as a reference. Although long-term colonization was not observed with any of the three strains, 16S rRNA gene profiling revealed that oral administration of DX_pv23 enhanced the recovery of antibiotic-disturbed microbiota to the original configuration significantly better than the other strains. The findings suggest that selected strains from FMT donors, such as DX_pv23 in this study, may have therapeutic potential by in vitro expression of colonization factors and boosting endogenous gut microbiota.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Impaired immunity and high attack rates caused by SARS-CoV-2 variants among vaccinated long-term care facility residents.

INTRODUCTION: Long-term care facilities (LTCF) residents are at high risk for severe coronavirus disease 2019 (COVID-19), and therefore, COVID-19 vaccinations were prioritized for residents and personnel in Finland at the beginning of 2021. METHODS: We investigated COVID-19 outbreaks in two LTCFs, where residents were once or twice vaccinated. After the outbreaks we measured immunoglobulin G (IgG) antibodies to severe acute respiratory syndrome coronavirus 2 spike glycoprotein, neutralizing antibody (NAb) titers, and cell-mediated immunity markers from residents and healthcare workers (HCWs). RESULTS: In LTFC-1, the outbreak was caused by an Alpha variant (B.1.1.7) and the attack rate (AR) among once vaccinated residents was 23%. In LTCF-2 the outbreak was caused by a Beta variant (B.1.351). Its AR was 47% although all residents had received their second dose 1 month before the outbreak. We observed that vaccination had induced lower IgG concentrations, NAb titers and cell-mediated immune responses in residents compared to HCWs. Only 1/8 residents had NAb to the Beta variant after two vaccine doses. CONCLUSIONS: The vaccinated elderly remain susceptible to breakthrough infections caused by Alpha and Beta variants. The weaker vaccine response in the elderly needs to be addressed in vaccination protocols, while new variants capable of evading vaccine-induced immunity continue to emerge.

Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant.

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

Urinary Excretion of Iohexol as a Permeability Marker in a Mouse Model of Intestinal Inflammation: Time Course, Performance and Welfare Considerations.

Intestinal permeability (IP) tests are used to assess intestinal damage in patients and research models. The probe iohexol has shown advantages compared to 51Cr-EDTA or absorbable/nonabsorbable sugars. During IP tests, animals are housed in metabolic cages (MCs) to collect urine. We examined the performance of an iohexol IP test in mice. Rag1-/- (C57BL/6) mice of both sexes were divided into controls or treatment groups, the latter receiving injections of effector/memory T cells to induce intestinal inflammation. After two, four and five weeks (W), a single dose of iohexol was orally administered. Urine was collected seven times over 24 h in MCs. Iohexol concentration was measured by ELISA. Intestinal histological damage was scored in duodenal sections. In control and treated mice of both sexes, urinary excretion of iohexol peaked at 4 h. From W2 to W4/W5, urinary iohexol excretion increased in treated mice of both sexes, consistent with development of duodenitis in this model. Positive correlations were observed between the urinary excretion of iohexol in W4/W5 and the histological severity of duodenitis in treated male mice. We conclude that a 6 h cumulative urine sample appears sufficient to evaluate small IP to iohexol in this mouse model, improving animal welfare by reducing cage periods.

No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children.

Narcolepsy type 1, likely an immune-mediated disease, is characterized by excessive daytime sleepiness and cataplexy. The disease is strongly associated with human leukocyte antigen (HLA) DQB1∗06:02. A significant increase in the incidence of childhood and adolescent narcolepsy was observed after a vaccination campaign with AS03-adjuvanted Pandemrix influenza vaccine in Nordic and several other countries in 2010 and 2011. Previously, it has been suggested that a surface-exposed region of influenza A nucleoprotein, a structural component of the Pandemrix vaccine, shares amino acid residues with the first extracellular domain of the human OX2 orexin/hypocretin receptor eliciting the development of autoantibodies. Here, we analyzed, whether H1N1pdm09 infection or Pandemrix vaccination contributed to the development of autoantibodies to the orexin precursor protein or the OX1 or OX2 receptors. The analysis was based on the presence or absence of autoantibody responses against analyzed proteins. Entire OX1 and OX2 receptors or just their extracellular N-termini were transiently expressed in HuH7 cells to determine specific antibody responses in human sera. Based on our immunofluorescence analysis, none of the 56 Pandemrix-vaccinated narcoleptic patients, 28 patients who suffered from a laboratory-confirmed H1N1pdm09 infection or 19 Pandemrix-vaccinated controls showed specific autoantibody responses to prepro-orexin, orexin receptors or the isolated extracellular N-termini of orexin receptors. We also did not find any evidence for cell-mediated immunity against the N-terminal epitopes of OX2. Our findings do not support the hypothesis that the surface-exposed region of the influenza nucleoprotein A would elicit the development of an immune response against orexin receptors.

Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease.

BACKGROUND & AIMS: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. METHODS: We performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. RESULTS: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. CONCLUSIONS: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.

Minor Effect of Antibiotic Pre-treatment on the Engraftment of Donor Microbiota in Fecal Transplantation in Mice.

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI) and is also considered a potential treatment for a wide range of intestinal and systemic diseases. FMT corrects the microbial dysbiosis associated with rCDI, and the engraftment of donor microbiota is likely to play a key role in treatment efficacy. For disease indications other than rCDI, FMT treatment efficacy has been moderate. This may be partly due to stronger resilience of resident host microbiota in patients who do not suffer from rCDI. In rCDI, patients typically have undergone several antibiotic treatments prior to FMT, depleting the microbiota. In this study, we addressed the effect of broad-spectrum antibiotics (Ab) as a pre-treatment to FMT on the engraftment of donor microbiota in recipients. We conducted a pre-clinical study of FMT between two healthy mouse strains, Balb/c as donors and C57BL/6 as recipients, to perform FMT within the same species and to mimic interindividual FMT between human donors and patients. Microbiota composition was assessed with high-throughput 16S rDNA amplicon sequencing. The microbiota of Balb/c and C57BL/6 mice differed significantly, which allowed for the assessment of microbiota transplantation from the donor strain to the recipient. Our results showed that Ab-treatment depleted microbiota in C57BL/6 recipient mice prior to FMT. The diversity of microbiota did not recover spontaneously to baseline levels during 8 weeks after Ab-treatment, but was restored already at 2 weeks in mice receiving FMT. Interestingly, pre-treatment with antibiotics prior to FMT did not increase the overall similarity of the recipient's microbiota to that of the donor's, as compared with mice receiving FMT without Ab-treatment. Pre-treatment with Ab improved the establishment of only a few donor-derived taxa, such as Bifidobacterium, in the recipients, thus having a minor effect on the engraftment of donor microbiota in FMT. In conclusion, pre-treatment with broad-spectrum antibiotics did not improve the overall engraftment of donor microbiota, but did improve the engraftment of specific taxa. These results may inform future therapeutic studies of FMT.

Deep sequencing of blood and gut T-cell receptor ß-chains reveals gluten-induced immune signatures in celiac disease.

Celiac disease (CD) patients mount an abnormal immune response to gluten. T-cell receptor (TCR) repertoires directed to some immunodominant gluten peptides have previously been described, but the global immune response to in vivo gluten exposure in CD has not been systematically investigated yet. Here, we characterized signatures associated with gluten directed immune activity and identified gluten-induced T-cell clonotypes from total blood and gut TCR repertoires in an unbiased manner using immunosequencing. CD patient total TCR repertoires showed increased overlap and substantially altered TRBV-gene usage in both blood and gut samples, and increased diversity in the gut during gluten exposure. Using differential abundance analysis, we identified gluten-induced clonotypes in each patient that were composed of a large private and an important public component. Hierarchical clustering of public clonotypes associated with dietary gluten exposure identified subsets of highly similar clonotypes, the most proliferative of which showing significant enrichment for the motif ASS[LF]R[SW][TD][DT][TE][QA][YF] in PBMC repertoires. These results show that CD-associated clonotypes can be identified and that common gluten associated immune response features can be characterized in vivo from total repertoires, with potential use in disease stratification and monitoring.

Autoantibodies against ganglioside GM3 are associated with narcolepsy-cataplexy developing after Pandemrix vaccination against 2009 pandemic H1N1 type influenza virus.

Following the mass vaccinations against pandemic influenza A/H1N1 virus in 2009, a sudden increase in juvenile onset narcolepsy with cataplexy (NC) was detected in several European countries where AS03-adjuvanted Pandemrix vaccine had been used. NC is a chronic neurological disorder characterized by excessive daytime sleepiness and cataplexy. In human NC, the hypocretin-producing neurons in the hypothalamus or the hypocretin signaling pathway are destroyed by an autoimmune reaction. Both genetic (e.g. HLA-DQB1*0602) and environmental risk factors (e.g. Pandemrix) contribute to the disease development, but the underlying and the mediating immunological mechanisms are largely unknown. Influenza virus hemagglutinin is known to bind gangliosides, which serve as host cell virus receptors. Anti-ganglioside antibodies have previously been linked to various neurological disorders, like the Guillain-Barré syndrome which may develop after infection or vaccination. Because of these links we screened sera of NC patients and controls for IgG anti-ganglioside antibodies against 11 human brain gangliosides (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b) and a sulfatide by using a line blot assay. Samples from 173 children and adolescents were analyzed: 48 with Pandemrix-associated NC, 20 with NC without Pandemrix association, 57 Pandemrix-vaccinated and 48 unvaccinated healthy children. We found that patients with Pandemrix-associated NC had more frequently (14.6%) anti-GM3 antibodies than vaccinated healthy controls (3.5%) (P = 0.047). Anti-GM3 antibodies were significantly associated with HLA-DQB1*0602 (P = 0.016) both in vaccinated NC patients and controls. In general, anti-ganglioside antibodies were more frequent in vaccinated (18.1%) than in unvaccinated (7.3%) individuals (P = 0.035). Our data suggest that autoimmunity against GM3 is a feature of Pandemrix-associated NC and that autoantibodies against gangliosides were induced by Pandemrix vaccination.

Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk.

BACKGROUND: Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated. METHODS AND FINDINGS: Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06:02 risk allele and in DQB1*06:02 transgenic mice immunized with Pandemrix when compared to controls. CONCLUSIONS: This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06:02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.

Testing safety of germinated rye sourdough in a celiac disease model based on the adoptive transfer of prolamin-primed memory T cells into lymphopenic mice.

UNLABELLED: The current treatment for celiac disease is strict gluten-free diet. Technical processing may render gluten-containing foods safe for consumption by celiac patients, but so far in vivo safety testing can only be performed on patients. We modified a celiac disease mouse model to test antigenicity and inflammatory effects of germinated rye sourdough, a food product characterized by extensive prolamin hydrolysis. Lymphopenic Rag1-/- or nude mice were injected with splenic CD4+CD62L-CD44high-memory T cells from gliadin- or secalin-immunized wild-type donor mice. We found that: 1) Rag1-/- recipients challenged with wheat or rye gluten lost more body weight and developed more severe histological duodenitis than mice on gluten-free diet. This correlated with increased secretion of IFNγ, IL-2, and IL-17 by secalin-restimulated splenocytes. 2) In vitro gluten testing using competitive R5 ELISA demonstrated extensive degradation of the gluten R5 epitope in germinated rye sourdough. 3) However, in nude recipients challenged with germinated rye sourdough (vs. native rye sourdough), serum anti-secalin IgG/CD4+ T helper 1-associated IgG2c titers were only reduced, but not eliminated. In addition, there were no reductions in body weight loss, histological duodenitis, or T cell cytokine secretion in Rag1-/- recipients challenged accordingly. IN CONCLUSION: 1) prolamin-primed CD4+CD62L-CD44high-memory T cells induce gluten-sensitive enteropathy in Rag1-/- mice. 2) Hydrolysis of secalins in germinated rye sourdough remains incomplete. Secalin peptides retain B and T cell stimulatory capacity and remain harmful to the intestinal mucosa in this celiac disease model. 3) Current antibody-based prolamin detection methods may fail to detect antigenic gluten fragments in processed cereal food products.

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4.

Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4-MD2-CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients' biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.

BACKGROUND & AIMS: The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice. METHODS: Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction. RESULTS: Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated. CONCLUSIONS: TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.

Human risk allele HLA-DRB1*0405 predisposes class II transgenic Ab0 NOD mice to autoimmune pancreatitis.

BACKGROUND & AIMS: Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice. METHODS: CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice. RESULTS: CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity. CONCLUSIONS: Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP.