A gut bacterial signature in blood and liver tissue characterizes cirrhosis and hepatocellular carcinoma.

BACKGROUND: HCC is the leading cause of cancer in chronic liver disease. A growing body of experimental mouse models supports the notion that gut-resident and liver-resident microbes control hepatic immune responses and, thereby, crucially contribute to liver tumorigenesis. However, a comprehensive characterization of the intestinal microbiome in fueling the transition from chronic liver disease to HCC in humans is currently missing. METHODS: Here, we profiled the fecal, blood, and liver tissue microbiome of patients with HCC by 16S rRNA sequencing and compared profiles to nonmalignant cirrhotic and noncirrhotic NAFLD patients. RESULTS: We report a distinct bacterial profile, defined from 16S rRNA gene sequences, with reduced α-and ß-diversity in the feces of patients with HCC and cirrhosis compared to NAFLD. Patients with HCC and cirrhosis exhibited an increased proportion of fecal bacterial gene signatures in the blood and liver compared to NAFLD. Differential analysis of the relative abundance of bacterial genera identified an increased abundance of Ruminococcaceae and Bacteroidaceae in blood and liver tissue from both HCC and cirrhosis patients compared to NAFLD. Fecal samples from cirrhosis and HCC patients both showed a reduced abundance for several taxa, including short-chain fatty acid-producing genera, such as Blautia and Agathobacter. Using paired 16S rRNA and transcriptome sequencing, we identified a direct association between gut bacterial genus abundance and host transcriptome response within the liver tissue. CONCLUSIONS: Our study indicates perturbations of the intestinal and liver-resident microbiome as a critical determinant of patients with cirrhosis and HCC.

B and T Cell Responses to SARS-CoV-2 Vaccination in Kidney and Liver Transplant Recipients with and without Previous COVID-19.

(1) Background: Vulnerable populations including transplant recipients are jeopardised by COVID-19. Herein, we report on B and T cell responses among liver and kidney organ recipients at our centre. (2) Methods: 23 liver and 45 kidney (14 thereof combined kidney/pancreas) transplanted patients were vaccinated with two doses of BNT162b2 followed by a booster dose of mRNA-1273 in 28 non-responders 4 months thereafter. Anti-SARS-CoV-2-Ig was measured by specific ELISA and virus neutralisation assay; T cell responses were measured by a spike protein-specific IFN-γ release assay. (3) Results: Compared to controls, B and T cell responses were weak in transplant recipients, particularly in those without prior exposure to SARS-CoV-2. Within this group, only 15% after the first and 58.3% after the second vaccination achieved seroconversion. A total of 14 out of 28 vaccination non-responders achieved a seroconversion after a third dose. Vaccination side effects were more frequent in healthy controls. The use of mycophenolate was associated with reduced anti-SARS-CoV-2-Ig production. (4) Conclusions: Our data confirm that vaccination responses are insufficient after standard vaccination in liver and kidney transplant recipients and are affected to a variable degree by specific immunosuppressants, particularly mycophenolate. Monitoring vaccination success and re-vaccinating those who are unresponsive seems prudent to achieve sufficient titres. Overall, prospective large-scale, multinational, multicentre studies or high-quality meta-analyses will be needed to generate personalised vaccination strategies in order to achieve protective immunity in high-risk, hard-to-immunize populations.

Short- and Long-Term Effects of a Prebiotic Intervention with Polyphenols Extracted from European Black Elderberry-Sustained Expansion of Akkermansia spp.

(1) Background: The intestinal microbiome has emerged as a central factor in human physiology and its alteration has been associated with disease. Therefore, great hopes are placed in microbiota-modulating strategies. Among various approaches, prebiotics, substrates with selective metabolization conferring a health benefit to the host, are promising candidates. Herein, we studied the prebiotic properties of a purified extract from European black elderberries, with a high and standardized content of polyphenols and anthocyanins. (2) Methods: The ELDERGUT trial represents a 9-week longitudinal intervention study divided into 3 distinct phases, namely a baseline, an intervention and a washout period, three weeks each. The intervention consisted of capsules containing 300 mg elderberry extract taken twice a day. Patient-reported outcomes and biosamples were collected weekly. Microbiome composition was assessed using 16S amplicon metagenomics. (3) Results: The supplementation was well tolerated. Microbiome trajectories were highly individualized with a profound shift in diversity indices immediately upon initiation and after termination of the compound. This was accompanied by corresponding changes in species abundance over time. Of particular interest, the relative abundance of Akkermansia spp. continued to increase in a subset of participants even beyond the supplementation period. Associations with participant metadata were detected.

Postacute COVID-19 is Characterized by Gut Viral Antigen Persistence in Inflammatory Bowel Diseases.

BACKGROUND & AIMS: The coronavirus disease 2019 (COVID-19) pandemic has affected populations, societies, and lives for more than 2 years. Long-term sequelae of COVID-19, collectively termed the postacute COVID-19 syndrome, are rapidly emerging across the globe. Here, we investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen persistence underlies the postacute COVID-19 syndrome. METHODS: We performed an endoscopy study with 46 patients with inflammatory bowel disease (IBD) 219 days (range, 94-257) after a confirmed COVID-19 infection. SARS-CoV-2 antigen persistence was assessed in the small and large intestine using quantitative polymerase chain reaction of 4 viral transcripts, immunofluorescence of viral nucleocapsid, and virus cultivation from biopsy tissue. Postacute COVID-19 was assessed using a standardized questionnaire, and a systemic SARS-CoV-2 immune response was evaluated using flow cytometry and enzyme-linked immunosorbent assay at endoscopy. IBD activity was evaluated using clinical, biochemical, and endoscopic means. RESULTS: We report expression of SARS-CoV-2 RNA in the gut mucosa ∼7 months after mild acute COVID-19 in 32 of 46 patients with IBD. Viral nucleocapsid protein persisted in 24 of 46 patients in gut epithelium and CD8+ T cells. Expression of SARS-CoV-2 antigens was not detectable in stool and viral antigen persistence was unrelated to severity of acute COVID-19, immunosuppressive therapy, and gut inflammation. We were unable to culture SARS-CoV-2 from gut tissue of patients with viral antigen persistence. Postacute sequelae of COVID-19 were reported from the majority of patients with viral antigen persistence, but not from patients without viral antigen persistence. CONCLUSION: Our results indicate that SARS-CoV-2 antigen persistence in infected tissues serves as a basis for postacute COVID-19. The concept that viral antigen persistence instigates immune perturbation and postacute COVID-19 requires validation in controlled clinical trials.

Calibrated comparison of SARS-CoV-2 neutralizing antibody levels in response to protein-, mRNA-, and vector-based COVID-19 vaccines.

SARS-CoV-2 neutralizing antibodies have been suggested to reflect the efficacy of COVID-19 vaccines. This study reports the direct comparison of the SARS-CoV-2 neutralizing antibody response elicited by a protein- (NVX-CoV2373), an mRNA- (Comirnaty), and a vector-based (Vaxzevria) COVID-19 vaccine, calibrated against the WHO international SARS-CoV-2 antibody standard, and further supports the use of neutralizing antibody levels as a correlate of protection.

Tofacitinib-Induced Modulation of Intestinal Adaptive and Innate Immunity and Factors Driving Cellular and Systemic Pharmacokinetics.

OBJECTIVE: By interfering with multiple cytokines, human Janus kinase inhibitors (JAKis) are of growing importance in the treatment of malignant and inflammatory conditions. Although tofacitinib has demonstrated efficacy as the first-in-class JAKi in ulcerative colitis many aspects concerning its mode of action and pharmacokinetics remain unresolved. DESIGN: We studied tofacitinib's impact on various primary human innate and adaptive immune cells. In-depth in vivo studies were performed in dextran sodium sulfate-induced colitis in mice. Immune populations were characterized by flow cytometry and critical transcription factors and effector cytokines were analyzed. Pharmacokinetics of tofacitinib was studied by liquid chromatography-tandem mass spectrometry. RESULTS: Tofacitinib inhibited proliferation in CD4+ and CD8+ T cells along with Th1 and Th17 differentiation, while Th2 and regulatory T cell lineages were largely unaffected. Monocytes and macrophages were directed toward an anti-inflammatory phenotype and cytokine production was suppressed in intestinal epithelial cells. These findings were largely reproducible in murine cells of the inflamed mucosa in dextran sulfate sodium colitis. Short-term treatment with tofacitinib had little impact on the mouse microbiota. Strikingly, the degree of inflammation and circulating tofacitinib levels showed a strong positive correlation. Finally, we identified inflammation-induced equilibrative nucleoside transporters as regulators of tofacitinib uptake into leukocytes. CONCLUSIONS: We provide a detailed analysis of the cell-specific immune-suppressive effects of the JAKis tofacitinib on innate and adaptive immunity and reveal that intestinal inflammation critically impacts tofacitinib's pharmacokinetics in mice. Furthermore, we describe an unappreciated mechanism-namely induction of equilibrative nucleoside transporters-enhancing baseline cellular uptake that can be inhibited pharmaceutically.

B and T cell response to SARS-CoV-2 vaccination in health care professionals with and without previous COVID-19.

BACKGROUND: In recent months numerous health care professional acquired COVID-19 at the workplace resulting in significant shortages in medical and nursing staff. We investigated how prior COVID-19 affects SARS-CoV-2 vaccination and how such knowledge could facilitate frugal vaccination strategies. METHODS: In a cohort of 41 healthcare professionals with (n=14) and without (n=27) previous SARS-CoV-2 infection, we assessed the immune status before, during and after vaccination with BNT162b2. The humoral immune response was assessed by receptor binding domain ELISA and different SARS-CoV-2 neutralisation assays using wildtype and pseudo-typed viruses. T cell immunity against SARS-CoV-2 surface and nucleocapsid peptides were studied using interferon-γ release assays and intracellular flow cytometry. Vaccine-related side effects were captured. FINDINGS: Prior COVID-19 resulted in improved vaccine responses both in the B and T cell compartment. In vaccine recipients with prior COVID-19, the first vaccine dose induced high antibody concentrations comparable to seronegative vaccine recipients after two injections. This translated into more efficient neutralisation of virus particles, even more pronounced than expected from the RBD ELISA results. Furthermore, T cell responses were stronger in convalescents and particularly strong against the SARS-CoV-2 nucleocapsid protein. INTERPRETATION: Herein, we corroborate recent findings suggesting that in convalescents a single vaccine dose is sufficient to boost adequate in vitro neutralisation of SARS-CoV-2 and therefore may be sufficient to induce adequate protection against severe COVID-19. New spike mutated virus variants render the highly conserved nucleocapsid protein - eliciting strong SARS-CoV-2 specific T cell immunity - an interesting additional vaccine target. FUNDING: Christian Doppler Research Association, Johannes Kepler University Linz.

Faecal Biomarkers in Inflammatory Bowel Diseases: Calprotectin Versus Lipocalin-2-a Comparative Study.

BACKGROUND AND AIMS: Faecal biomarkers, particularly calprotectin [FCAL], have become important diagnostic and monitoring tools in inflammatory bowel diseases [IBD]. As FCAL is mainly produced by neutrophils, we hypothesised that faecal lipocalin-2 [FLCN2], also expressed by intestinal epithelial cells [IEC], could be beneficial in specific clinical situations. METHODS: We compared clinical and endoscopic activity-related correlations between FCAL and FLCN2, assayed from the same sample, in a cohort of 132 patients (72 Crohn's disease [CD]) and 40 controls. A detailed analysis of cellular origins was done by confocal microscopy and flow cytometry. To evaluate the potential to detect low-grade inflammation, we studied faecal and tissue concentrations in a cohort with clinical, endoscopic, and histological remission. RESULTS: There was an excellent correlation between FCAL and FLCN2 [rS = 0.87, p <0.001] and comparable sensitivity and specificity to predict clinical and endoscopic disease activity, with optimal thresholds for endoscopic activity of 73.4 and 1.98 µg/g in ulcerative colitis [UC] and 78.4 and 0.56 µg/g in Crohn's disease for FCAL and FLCN2, respectively. Strong co-expression of both proteins was observed in granulocytes and macrophages. IECs expressed LCN2 but not CAL. In our IBD cohort in deep remission neither FCAL nor FLCN2 was different from controls; yet mucosal LCN2 but not CAL expressions remained elevated in the rectum of UC and the ileum of CD patients. CONCLUSIONS: This study corroborates the diagnostic equivalence of FLCN2 and FCAL in IBD. In remission, persistent mucosal overexpression renders LCN2 an attractive candidate for molecular inflammation warranting further investigation.