Introduction

Helicobacter pylori (H. pylori) infection is a major cause of gastric diseases, with the virulence factor CagA carried by exosomes playing a crucial role in regulating inflammation. Ferroptosis and M1 polarization are key mechanisms in promoting inflammation, yet the role of CagA in inducing macrophage ferroptosis and M1 polarization to exacerbate gastric mucosal inflammation remains unclear.

Aims & Methods

CagA-positive (CagA+) and CagA-negative (CagA-) H. pylori strains were co-cultured with gastric epithelial cells, and their supernatants were collected. Exosomes were isolated using ultracentrifugation to obtain CagA+Exo and CagA-Exo. Changes in macrophage M1/M2 polarization markers and ferroptosis markers were analyzed under CagA+Exo and CagA-Exo treatment. The activation of the JAK/STAT1 signaling pathway was assessed by Western blot, and STAT1 expression in the nucleus was evaluated using a nuclear protein extraction kit. Chromatin immunoprecipitation (ChIP) was used to identify the binding site of STAT1 on the SLC7A11 promoter. Biopsy samples from the gastric mucosa of gastritis patients infected with CagA+ or CagA- H. pylori were analyzed for macrophage polarization and ferroptosis markers.

Results

Exosomal CagA was found to induce macrophage M1 polarization and ferroptosis, with the ferroptosis inhibitor SLC7A11 playing a key role in CagA-induced ferroptosis. Exosomal CagA activated the JAK/STAT1 signaling pathway, leading to M1 polarization and ferroptosis. Additionally, CagA+Exo increased nuclear STAT1 expression, which subsequently bound to the SLC7A11 promoter to regulate its transcription. In gastric mucosa biopsies from CagA+ H. pylori-infected gastritis patients, increased levels of iNOS (a marker of M1 polarization) and decreased expression of SLC7A11 were observed.

Conclusion

Our findings indicate that exosomal CagA promotes macrophage M1 polarization and ferroptosis through the JAK/STAT1 signaling pathway. This study provides new insights and potential therapeutic targets for H. pylori-associated gastritis.

Introduction

Gastric mucosal visibility is essential for high-quality upper gastrointestinal (GI) endoscopy. While simethicone and N-acetylcysteine (NAC) have been shown to improve mucosal visibility, the optimal time interval between preparation and endoscopy remains undefined.

Aims & Methods

This randomized controlled trial evaluated the effect of timing on mucosal visibility and explored the upper limit beyond which visibility begins to decline.
In this single-center, randomized controlled trial, adult patients undergoing elective upper GI endoscopy were randomized into four groups: placebo, simethicone + NAC with endoscopy performed after 10–20 minutes, 20–30 minutes, and >30 minutes. The primary outcome was the proportion of patients achieving adequate gastric mucosal visibility (GMV), defined as a score of <7 on a validated visibility scale. A pre-specified subgroup analysis of the >30 minute group was performed by stratifying patients into 20-minute intervals (31–50, 51–70, and 71–90 minutes) to assess the durability of mucosal clarity over time.

Results

Comparison	Adequate gastric mucosal visibility (%)	P
10–20 min vs 20–30 min	27% vs 62%	0.001
10–20 min vs >30 min	27% vs 67%	0.001
10–20 min vs Placebo	27% vs 2.6%	0.001
20–30 min vs >30 min	62% vs 67%	0.174
20–30 min vs Placebo	62% vs 2.6%	0.001

1,140 patients (median age 42years, 63.2% males) were included in the study. Adequate GMV was significantly higher in all preparation groups compared to placebo (p < 0.001). The 20–30 minute and >30 minute groups demonstrated the highest rates of adequate visibility (61.5% and 73.5%, respectively), both significantly superior to the 10–20 minute group (26.9%) and placebo (2.6%). In the >30 minute subgroup analysis, the 31–50 minute interval demonstrated the highest adequacy (73.5%). The 51–70 minute group showed moderate adequacy (52.1%), which was not significantly different from the 20–30 minute group (p = 0.207). However, the 71–90 minute group had reduced adequacy (43.8%) and did not differ significantly from the 10–20 minute group (p = 0.145). No adverse events were reported across study arms.

Conclusion

Endoscopy performed between 20 and 50 minutes after mucosal preparation yields optimal gastric mucosal visibility. A decline in visibility beyond 50 minutes suggests a practical upper limit for endoscopy timing following preparation. These findings support structured, time-sensitive scheduling following mucosal cleansing to enhance procedural quality.

Disclosure

None

Introduction

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by dysregulated T-cell responses, causing chronic intestinal inflammation.¹ While anti-TNF therapy is established in IBD, targeting additional pathways may improve outcomes for patients who do not respond adequately to current treatments.² OX40L, an immune co-stimulator, has been implicated in the pathogenesis of several inflammatory conditions.³ Our study provides a comprehensive analysis of the effects of combined TNF and OX40L blockade in a murine adoptive T cell transfer (ATC) model of colitis, examining body weight, detailed histopathological parameters, and a broad range of inflammatory cytokines in colon tissue.

Aims & Methods

Colitis was induced in severe combined immunodeficiency (SCID) mice by intraperitoneal transfer of CD4⁺CD45RB^high T cells isolated from healthy BALB/c donor mice. We established five treatment groups (n=8-10): 1) CD45RB^low transfer “healthy control”, 2) CD45RB^high transfer with isotype antibodies “disease control group”, 3) CD45RB^high transfer with anti-TNF antibody (XT3.11, 250 μg/mouse), 4) CD45RB^high transfer with anti-OX40L antibody (RM134L, 250 μg/mouse), and 5) CD45RB^high transfer with both antibodies at 250 µg/mouse, each. Body weight was monitored for 46 days as a measure of disease progression and normalized to baseline weight at day 1. At study termination, we collected colon tissue for analysis of inflammatory cytokines using multiplex immunoassays and conducted histopathological examination. Histopathological scoring was performed blinded using a validated scoring system for colonic inflammation.⁴

Results

Overall, combined anti-TNF/anti-OX40L therapy was most effective in preventing body weight loss compared to the isotype disease control group, followed by anti-TNF monotherapy and anti-OX40L monotherapy (p<0.0001, p=0.0009, and p=0.03, respectively). Mean normalized body weight (MNBW) of the CD45RB^low healthy control group at termination on day 46 was 115.2 %, while CD45RB^high cell transfer in the disease control group triggered disease induction and body weight loss, with a MNBW of 93.8 % at day 46. The CD45RB^high groups treated with anti-TNF or anti OX40L monotherapy reached a MNBW of 107.6 % and 102.9%, respectively. The CD45RB^high group treated with combination therapy reached 112.6%, closest to the CD45RB^low healthy control group. Notably, only combination therapy led to a significant improvement in histopathological scoring (p=0.0176) compared to the isotype disease control. Similarly, combination treatment consistently resulted in the strongest downregulation of several inflammatory cytokines including IFNg, IL-4, or TNFa, which are increased in colon tissue in the CD45RB^high disease group compared to the CD45RB^low healthy control group.

Conclusion

Combined blockade of TNF and OX40L demonstrated significantly greater efficacy in the ATC preclinical model of IBD in prevention of weight loss, downregulation of key inflammatory cytokines, and reduction of histological damage compared to either monotherapy. These data suggest potential additive efficacy for TNF and OX40L inhibition in controlling intestinal inflammation, and warrant further exploration of dual TNF/OX40L inhibition in clinical trials, particularly among those with inadequate response to anti-TNF monotherapy.

References

1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol. 2010;28:573-621.
2. Vermeire S. Combination biologic therapy for ulcerative colitis. Lancet Gastroenterol Hepatol. 2023;8:288-90.
3. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10:414-33.
4. Erben U, Loddenkemper C, Doerfel K, Spieckermann S, Haller D, Heimesaat MM, Zeitz M, Siegmund B, Kühl AA. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7:4557-76.

Disclosure

Thomas Leeuw, Rolf Keiffer, Danping Ding-Pfennigdorff, Christian Asbrand, Raaj Mehta, Matthias Herrmann are employees of Sanofi and may hold stock or equity options in Sanofi