Introduction

Chronic atrophic gastritis (CAG) is primarily caused by Helicobacter pylori (Hp) infection but also by autoimmune phenomena giving rise to autoimmune atrophic gastritis (AAG), whose hallmark is corpus-restricted atrophy sparing the antrum (unlike Hp infection causing extensive CAG). AAG triggered by Hp has been proposed, but diagnosis of Hp in this condition is difficult. Hp infection is one of the main risk factors for gastric cancer (GC), while the role of AAG in the onset of GC is still debated. Assessing the association of GC with AAG is thus challenging, as it requires the exclusion of Hp infection. Hence, investigating the presence of Hp-related genes in the gastric mucosa of histologically Hp negative AAG patients complicated by GC could be useful for accurately assessing the role of Hp in GC associated with AAG.

Aims & Methods

The aim was to assess the presence of Hp-related genes in corpus mucosa biopsies using Real-time and Digital PCR in AAG patients with GC and negative Hp at histology, along with Hp-positive and -negative controls.
Single-centre, experimental molecular biology study derived from 713 pts with histological diagnosis of CAG (2009-2022). Pts without corpus-restricted CAG (n=118) and with histological positivity for Hp (n=57) were excluded, resulting a source population of 538 AAG pts with negative Hp at histology. Out of these, 10 pts (80%F, age 69.5 (range 50-83) yrs) were diagnosed with gastric neoplastic lesions (GNL), including gastric cancer (GC) and high-grade intraepithelial neoplasia (HG-IEN). Real-time and Digital PCR were used to assess the presence of Hp-related genes in corpus mucosa biopsies of the 10 GNL-AAG pts and 2 Hp-positive non-atrophic gastritis pts (positive controls: 1 F,1 M, age 39, 68 yrs) and 2 subjects with a healthy stomach (negative controls: 1 F,1 M, age 22, 34 yrs). DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections of corpus mucosa biopsies and Real-time and Digital PCR were performed using specific probes for UreA, UreC, and CagA Hp-related genes. 16sRNA and Beta2 microglobulin specific probes were used as amplification ctr of bacterial and human DNA. Real-Time PCR gives a semiquantitative analysis, whereas Digital PCR measures the accurate copy numbers of Hp DNA.

Results

GNL were diagnosed in 3/10 pts at AAG diagnosis, while in 7 at follow-up (median 96, range 12-156 mos). GNL were frequently in the corpus (50%), less commonly at incisura angularis (30%) or antrum (20%), and consisted of 8 GC and 2 HG-IEN. None had family history of GC, all pts were parietal cell autoantibodies positive. ELISA Hp serology was negative in all 5/10 available sera. Hp-related genes were not detected in any of the GNL-AAG pts nor in negative controls, but UreA and UreC were found in both positive controls and CagA in one. 16sRNA gene was detected in GNL-AAG by Real-time(CT mean 26.0, SD 0.3) and Digital PCR (copies μl mean 1397,SD 348) in similar amounts as in positive controls (mean CT 25.7, SD 0.9, mean copies/μl 4977.5, SD 2185.7), indicating the detectability of bacterial genes in the extracted DNA.

Conclusion

In FFPE corpus mucosa biopsies of pts with histologically Hp negative AAG and GC, specific Hp-related genes are detectable by nor Real-time neither Digital PCR. This result supports the idea that GC development cannot be excluded a priori in primary, Hp negative AAG, suggesting that Hp-unrelated cancerogenetic mechanisms may be involved and regular surveillance should be considered in primary AAG.

References

• Lenti MV et al. Autoimmune gastritis. Nat Rev Dis Primers. 2020.
• Lahner E et al. Chronic atrophic gastritis: Natural history, diagnosis and therapeutic management. A position paper. Dig Liver Dis. 2019.
• Rugge M et al; RE.GA.IN; RE GA IN. RE.GA.IN.: the Real-world Gastritis Initiative-updating the updates. Gut. 2024.
• Neumann WL et al. Autoimmune atrophic gastritis--pathogenesis, pathology and management. Nat Rev Gastroenterol Hepatol. 2013.

Introduction

Intestinal bacteria with 7α-dehydroxylating (7αDH) activity are able to produce the secondary bile acids, such as deoxycholic acid (DCA). Higher concentrations of DCA are linked to a higher risk of colorectal cancer (CRC) risk, yet underlying molecular mechanisms demonstrating a causal role of DCA are still unclear. This includes a recently demonstrated immunomodulatory effect of secondary bile acids, which may contribute to CRC pathogenesis. Here, we aim to investigate the tumor-promoting and immunomodulatory role of DCA in a gnotobiotic CRC mouse model.

Aims & Methods

Germ-free wildtype mice were colonized with a simplified bacterial consortium, the bile acid converting microbiota (BACOMI). BACOMI is able to produce DCA due to the 7αDH activity of Clostridium scindens (+7αDH). Control mice were colonized with BACOMI without C. scindens, lacking 7αDH activity (-7αDH) and DCA. A sub-group of mice was treated with AOM/DSS to induce colonic tumors. Bacterial abundances were measured in colonic content via qPCR, cecal bile acid composition was analyzed using LC-MS/MS and colonic tissue was analyzed using qPCR and immunofluorescence-staining. Primary T-cells and macrophages were isolated from the colon as well as mesenteric and iliac lymph nodes and analyzed by flow cytometry.

Results

DCA was exclusively present in mice colonized with the BACOMI consortium and led to higher colonic tumor numbers compared to mice colonized with BACOMI without C. scindens (+7αDH = 20.5 tumors±7.5 SD vs. -7αDH = 7.9 tumors±2.0 SD), in particular for small- and medium-sized tumors. Presence of DCA also resulted in shorter colon lengths and lower levels of butyrate in cecal content. In BACOMI colonized mice, the treatment with AOM/DSS promoted colonic colonization by C. scindens compared to mice without AOM/DSS treatment. Even without AOM/DSS treatment, the presence of C. scindens and DCA resulted in significantly higher numbers of Ki67+ epithelial cells in colonic crypts and more pronounced hypoxia in the colonic epithelium. Colonic expression of genes involved in CRC pathogenesis (e.g., Axin2, Ccnd1, Myc), bile acid metabolism (e.g., Nr1h4, Nr0b2, Slc51b) as well as immune responses (e.g., Nfkb1, Rorc, Itgam) was upregulated in mice with DCA. Sub-types of Foxp3+ regulatory T-cells were more abundant in mesenteric and iliac lymph nodes whereas numbers of Ly6C high macrophages were lower in the colon and mesenteric lymph nodes of mice with DCA compared to mice lacking DCA.

Conclusion

​In a gnotobiotic mouse model for CRC, the presence of microbially-derived DCA promotes colonic tumorigenesis in vivo. Even in the absence of chemically-induced tumorigenesis, DCA stimulates the expression of markers closely associated with CRC and promotes colonic epithelial proliferation. These findings strongly imply a causal role of DCA in the development of CRC. Additionally, DCA shows immunomodulatory effects, which may demonstrate another mechanistic link of how DCA affects intestinal health.

Introduction

Non-alcoholic fatty liver disease (NAFLD) often advances to more severe stages, namely steatohepatitis (NASH), and may further progress towards hepatocellular carcinoma (HCC). Still, the cellular and molecular mechanisms governing disease progression - and that may be ammendable to therapeutic intervention - remain elusive.

Aims & Methods

Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis.
Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n=199) or HCC (n=366 HCC and n=11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 weeks to induce NASH; and for 66 weeks to induce NASH-HCC.

Results

Results showed that hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile in obese patients. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice displayed increased peroxisome-proliferator activated receptor alpha (PPARalpha) protein levels, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis. Further, the pro-inflammatory/fibrogenic milieu was reduced. Mir21 KO mice presented with a lower number of pre-neoplastic nodules, impaired hepatocyte proliferation and downgraded oncogenic signaling, thus being protected from NASH-related carcinogenesis. The hsa-miRNA-21-5p/PPARalpha pathway was similarly deregulated in patients with HCC or NASH-related HCC, correlating with HCC markers and worse prognosis.

Conclusion

In conclusion, hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. Inhibition of hsa-miR-21-5p, leading to a pro-metabolomic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC (EXPL/MED-OUT/1317/2021, FCT, Portugal).

Introduction

A wide range of relationships between the microbial community and various environmental or individual factors have been proposed. Monozygotic and dizygotic twins provide a unique model for assessing host-microbial interactions. The mycobiome has been increasingly implicated in the development of several diseases, but knowledge of the dynamics of the fungal community, particularly in twins, has not been addressed.

Aims & Methods

In this work, we investigated the fungal community in a twin cohort and explored its relationship with associated genetic and environmental factors through in-depth analysis.
The twin cohort consisted of 106 well-characterised and systematically studied monozygotic and dizygotic pairs (212 stool samples). DNA was extracted and PCR reactions were performed to sequence the lower gastrointestinal microbiome and the mycobiome community. The bacterial community was characterised using the V1-V2 region of the 16 rRNA gene and the fungal community was analysed using the ITS1-ITS2 region.

Results

The fungal community, represented by the sum of all fungal sequences, showed a significantly higher heterogeneity than the bacterial community. Several individual and genetic factors, such as the zygotic status of the twins (monozygotic vs. dizygotic), as well as sex, mode of birth, breast-feeding as newborns or living in the same household, were independent of the total number of fungal sequences. The bacterial community was significantly more abundant than the fungal community, the most common representatives of which were Candida and Geotrichum with sometimes very high abundances. Saccharomyces was also common in the cohort, but only in low abundance, other fungi with a wider distribution were Cladosporium or Penicillium. The environmental and individual factors mentioned above were also correlated with the individual fungal genera, but there were no significant correlations found here either. The next step was to correlate the bacterial and fungal communities with each other, and strong negative correlations were found with certain bacteria such as Prevotella.

Conclusion

Genetic factors in twins are not associated with dynamics in the faecal fungal community and were the subject of high inter-individual variability. However, there is a strong association between bacterial and mycobial interaction, which requires further in-depth understanding of microbial interactions.

Disclosure

No conflict of interest to declare