René Gerolami

Background/Objectives: Systemic treatment of advanced hepatocellular carcinoma (HCC) is based on combinations of immunotherapies (ITs) and lacks predictive markers of efficacy. Objectives: To define the prognostic value of the CRAFITY and RECA biological scores for overall survival (OS) before and during IT, and to evaluate the value of these two models for predicting the therapeutic response. Patients and methods: This was a multicenter retrospective analysis of 229 patients. OS was analyzed using Kaplan–Meier curves, log-rank tests, and Cox models, through which second-line therapy was modeled as a time-dependent covariate to avoid immortal time bias. The predictive capacity was assessed using univariate logistic regression. Validation was performed within two external Chinese cohorts. Results: Sixty-six percent of patients had Barcelona Clinic Liver Cancer (BCLC) stage C HCC (vascular invasion: 36.3%, metastases: 32.6%). After a mean follow-up of 14.9 (12.8) months, the median OS was 17.4 (6.9–38.0) months. The CRAFITY score distinguished only two different prognostic subgroups before treatment, but its prognostic value was confirmed with three different prognostic groups after 3 and 5 cycles and 6 months of treatment. The RECA score was strongly associated with OS before treatment and after 3 and 5 cycles and after 6 months of IT. Conversely, neither score had a discriminatory ability to predict early therapeutic response. The prognostic value of both models for OS was confirmed in the external cohorts. Conclusions: The RECA and CRAFITY scores have strong prognostic value for OS during IT. Beyond the models, the dynamic effects of systemic inflammation on IT reveal distinct clinical outcomes. Neither score has the ability to predict early therapeutic response, further supporting their use during treatment.

Background: Hepatitis B virus (HBV) infection is a global health epidemic that causes fatal complications, leading to liver cirrhosis and hepatocellular carcinoma. The link between HBV-related dysbiosis and specific bacterial taxa is still under investigation. Enterocloster is emerging as a new genus (formerly Clostridium), including Enterocloster bolteae, a gut pathogen previously associated with dysbiosis and human diseases such as autism, multiple sclerosis, and inflammatory bowel diseases. Its role in liver diseases, especially HBV infection, is not reported. Methods: The fecal samples of eight patients with chronic HBV infection and ten healthy individuals were analyzed using the high-throughput culturomics approach and compared to 16S rRNA sequencing. Quantification of ethanol, known for its damaging effect on the liver, produced from bacterial strains enriched in chronic HBV was carried out by gas chromatography–mass spectrometry. Results: Using culturomics, 29,120 isolated colonies were analyzed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI–TOF); 340 species were identified (240 species in chronic HBV samples, 254 species in control samples) belonging to 169 genera and 6 phyla. In the chronic HBV group, 65 species were already known in the literature; 48 were associated with humans but had not been previously found in the gut, and 17 had never been associated with humans previously. Six species were newly isolated in our study. By comparing bacterial species frequency, three bacterial genera were serendipitously found with significantly enriched bacterial diversity in patients with chronic HBV: Enterocloster, Clostridium, and Streptococcus (p = 0.0016, p = 0.041, p = 0.053, respectively). However, metagenomics could not identify this enrichment, possibly concerning its insufficient taxonomical resolution (equivocal assignment of operational taxonomic units). At the species level, the significantly enriched species in the chronic HBV group almost all belonged to class Clostridia, such as Clostridium perfringens, Clostridium sporogenes, Enterocloster aldenensis, Enterocloster bolteae, Enterocloster clostridioformis, and Clostridium innocuum. Two E. bolteae strains, isolated from two patients with chronic HBV infection, showed high ethanol production (27 and 200 mM). Conclusions: Culturomics allowed us to identify Enterocloster species, specifically, E. bolteae, enriched in the gut microbiota of patients with chronic HBV. These species had never been isolated in chronic HBV infection before. Moreover, ethanol production by E. bolteae strains isolated from the chronic HBV group could contribute to liver disease progression. Additionally, culturomics might be critical for better elucidating the relationship between dysbiosis and chronic HBV infection in the future.

Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn). Candida yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05–1.43] vs. 0.034 [0.008–0.57], p = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, p = 0.0001). Pichia kudriavzevii was the most frequent (four patients), while Candida glabrata, Candida albicans, and Galactomyces geotrichum were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49–5.60] vs. 0.32 [0.009–0.43], p = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. Pichia kudriavzevii strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to K. pneumoniae, gut Pichia and Candida yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production.

Hepatitis E is mostly autochthonous in Western developed countries, eating pig-derived products being the most frequently documented source. Hepatitis E virus (HEV) infection is usually asymptomatic or self-limiting, but it can cause acute liver failure. HEV serological testing was performed using EUROIMMUN immunoenzymatic assays. HEV RNA in the serum was determined using an in-house real-time reverse transcriptase PCR procedure. The HEV genotype was determined through phylogenetic analysis after Sanger sequencing was performed using an in-house procedure. The case patient, an immunocompetent patient in his 60s with type 2 diabetes and no documented chronic liver disease, was hospitalized in February 2021 in an intensive care unit due to an initially unexplained coma. He presented metformin overdose and fulminant hepatitis E (HEV RNA in the serum was 4,140,000 copies/mL) that evolved toward death. The HEV genotype was 3f. We identified eight previous hepatitis E in diabetic patients, but with no metformin excessive plasma concentration, in the literature. Three patients were liver transplant recipients and three died. HEV infection can be severe and life-threatening in diabetic patients, which warrants HEV testing in this special population in the case of an altered general condition and/or liver cytolysis.

Linked ContentThis article is linked to Borentain and Colson et al paper. To view this article visit https://doi.org/10.1111/apt.14188.

Purpose Interactions between endothelial and tumor cells via E-selectin and sialyl Lewis x (sLex) have been suggested to play a significant role in the development of metastasis and tumor growth. In this work, we tested whether inhibition of E-selectin expression on the surface of endothelial cells might impair endothelial/tumor cells interactions and tumor growth of hepatocarcinoma cells in vitro and in vivo. Methods We used HepG2 cells that highly express sLex antigens and HuH7 cells that do not express sLex. Inhibition of E-selectin expression on the surface of endothelial cells was obtained by using cimetidine and amiloride treatment. Results Cimetidine and amiloride inhibited, respectively, by 20 and 64 % E-selectin expression by activated endothelial cells and significantly subsequent adhesion of HepG2 cells to activated endothelial cells. Subcutaneous injection of cimetidine or amiloride resulted in a significant inhibition of HepG2 cells tumor growth in nu/nu mice but not of HuH7 cells. Thus, cimetidine and amiloride administration led to an inhibition of 57 and 75 % of HepG2 tumor growth in vivo, respectively. This effect was associated with an inhibition of vasculogenesis as demonstrated by anti-CD31 immunostaining. Conclusion Inhibition of E-selectin expression allows an anti-tumoral effect on sLex-expressing HCC tumors in vivo. This suggests that interactions between HCC cells and endothelial cells through sLex antigens and E-selectin might be a target for treatment of HCC. Further studies might evaluate the clinical impact of cimetidine and amiloride in the treatment of HCC patients alone or in combination with other anti-tumoral agents.

Hepatitis B virus (HBV) infection is a public health problem. In France, 0.68% of adults are chronically infected. We aimed to describe the epidemiological, virological and clinical characteristics of HBV infections newly diagnosed in 2011 in University hospitals of Marseille, the second largest French city. HBV serology was performed for 18,130 sera from 15,744 patients. A total of 167 patients were newly-diagnosed with HBV based upon the detection of hepatitis B surface antigen and anti-hepatitis B core antibodies. Clinico-epidemiological features were analyzed for 78 patients. Patients included a majority of men (59%), women being significantly younger with a mean age of 36 ± 17 versus 43.5 ± 16.2 years (P = 0.009). Country of birth was available for 52 patients and 35% of them originated from sub-Saharan Africa. Levels of the liver biological parameters were significantly lower in women compared to men, in whom mean alanine aminotransferase and gammaglutamyl transferase levels were 24 ± 39 versus 37 ± 36 IU/l (P = 0.0001) and 20 ± 20 versus 51 ± 53 IU/l (P = 0.0001), respectively. Co-infections with hepatitis C and human immunodeficiency viruses were found in 5% and 6% of the patients, respectively. HBV DNA was detectable in 90% of the HBeAg-negative patients. In addition, there was a positive correlation between the HBsAg titer and the HBV DNA level (P = 0.001). Genotype D was the most common HBV genotype and was found in 53% of the patients tested, followed by genotype E (21%). HBV remains a major concern with a slightly greater number of new diagnoses than in 2004. HBV genetic diversity was substantial in the present cohort. J. Med. Virol. 88:828–836, 2016.