Marietta, Eric V.

Abstract Genome-resolved virome analysis remains inaccessible for many samples, including those with clinical relevance, because viral nucleic acid recovered after enrichment is often too scarce to support de novo genome assembly. As a result, many analyses are limited to sparse read-level detection, which cannot recover divergent viruses, resolve strains, or interpret gene-level variation. Here, we developed Low Biomass Viral Sequencing (LBV-Seq), a workflow that couples low-input viral sample handling with modified primary template-directed amplification and short- or long-read sequencing to enable de novo reconstruction of DNA and RNA viral genomes from sub-femtogram to nanogram inputs. LBV-Seq reproducibly captures the same relative community composition, amplifies diverse viruses, and achieved broad genome coverage across nearly all targets regardless of viral genome structure, Baltimore class, abundance, or input mass. Short-read assemblies recovered near-complete genomes from femtogram-scale inputs. Long-read sequencing provided orthogonal support for genome structure, with PacBio HiFi reads spanning large portions of viral genomes and, in some cases, complete small viral genomes. Applied to virus-enriched human duodenal biopsy eluates, LBV-Seq provided proof-of-feasibility for recovering both bacteriophage and eukaryotic viral genomes from low-input biopsy-derived material. In the eluates tested, LBV-Seq recovered co-occurring Alphatorquevirus and Betatorquevirus genomes estimated to be present at roughly 10 copies/µL and at viral masses below 0.1 fg/µL. LBV-Seq enables genome-resolved virome analysis in samples previously limited to detection-based viromics, supporting viral discovery and strain-resolved analyses in settings where viral mass is low, including viruses enriched from human tissue biopsies.

SummaryBackgroundThe Th2 allergic pathway in eosinophilic oesophagitis (EoE) responds to food antigen exposure.AimTo compare the presence and temporal pattern of food antigen penetration in oesophageal mucosa in active and inactive EoE and controlsMethodsThirty‐two patients with EoE (20 active) and 10 controls were asked to eliminate all wheat and/or dairy 12, 24, 48, 72 or 96 hours before endoscopy. Immunostaining on endoscopic biopsies was performed for gliadin, casein and whey.ResultsGluten, casein and whey were detected by positive staining in 17/32 (53.1%), 21/32 (65.6%), and 30/32 (92.0%) of patients, respectively. In active vs inactive EoE, 70.0% vs 25.0% (P < 0.05), 80.0% vs 41.5%, and 90.0% vs 90.9% patients had detectable gliadin, casein and whey, respectively. Casein and whey (20.0% and 100%, respectively) but not gliadin, were present in controls. The gliadin staining density was greater in active compared to inactive disease at ≤ 24 vs >24 hours after exposure (P = 0.05) but no differences were detected when comparing active and inactive patients for casein and whey. There was greater staining density for whey than casein for all patients at ≤24 hours (mean 2.14 ± 0.91 and 1.07 ± 1.33, P = 0.02). In active EoE, IgG4 was present in 14/20 compared to one inactive patient.ConclusionThe oesophageal epithelium is selectively permeable and has relatively long dwell times for food antigens known to trigger EoE. The precise mechanism of antigen‐specific mucosal entry and the factors that determine the induction or effector trigger of the Th2 pathway activation merit further study.

Background and aims: Clinical experience suggests that patients with coeliac disease (CD) are more prone to develop herpes zoster (HZ), but robust studies are lacking. Methods: We identified 29,064 patients with CD 1969−2008 using biopsy report data from Sweden’s 28 pathology departments. CD was equalled to villous atrophy (Marsh histopathology grade III). Each patient was matched on age, sex, calendar year and county of residence to up to five reference individuals ( n=144,342) from the general population. We then used Cox regression to estimate hazard ratios (HRs) for future HZ (defined as having a hospital-based inpatient or outpatient record of this diagnosis in the Swedish Patient Register). Results: During follow-up, 154 (0.53%) individuals with CD and 499 (0.35%) reference individuals developed HZ. Among individuals aged ≥60 years, 1.06% of CD individuals and 0.85% of reference individuals had a lifetime record of HZ. Overall, CD was associated with a 1.62-fold increased risk of HZ (95% CI=1.35−1.95), and was seen also when we considered comorbidity with lymphoproliferative disease, systemic lupus erythematosus, type 1 diabetes, thyroid disease, rheumatoid disease and excluded individuals with a record of dermatitis herpetiformis. The increased risk remained significant after more than five years of follow-up (1.46; 1.16−1.84) Conclusions: CD is associated with HZ, the increased relative risk persists over time from celiac diagnosis but the absolute risk is small.

ObjectiveThe gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut‐derived commensal to modulate immune response and treat arthritis in a humanized mouse model.MethodsWe isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis‐susceptible HLA–DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice.ResultsWhen treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr‐1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen‐specific Th17 responses and increased transcription of interleukin‐10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll‐like receptor 4 (TLR‐4) and TLR‐9 was not affected in treated mice.ConclusionOur results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

Abstract The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8+ T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.