Introduction

Fecal microbiota transplantation (FMT) is most effective treatment for recurrent clostridioides difficile infection (rCDI). Some factors associated with donor, recipients and FMT protocols (amount of feces, infusion route, number of infusion, time of thaw) affect FMT outcome. However, the appropriate FMT protocol is still being studied, and research on the most effective thawing time is lacking.

Aims & Methods

The purpose of this study was to optimize the effect of thawing time of frozen FMT stool on rCDI.
This prospective, single center and randomized controlled trial was performed from October 2022 to December 2023. Patients were randomly assigned to groups that received stool for FMT thawed 4 hours at room temperature (4HTG) or thawed 12 hours in a refrigerator(12HTG). Stools were donated from single healthy donor. Each donation was diluted in glycerol-saline solution (12.5% glycerol in 0.90% w/v NaCl in water) and thoroughly homogenized. A 250 mL formulation containing 60g of stool was stored at -80 °C and administered via colonoscopy within 4 months. The primary outcome was the cumulative recurrence rate of Clostridioides difficile-associated diarrhea (CDAD) at week 8. The CDAD resolution rate at 1 week, adverse events related to thawing time were also evaluated.

Results

Total of 22 patients were randomized into 4HTG (n=12) vs 12HTG (n=10). CDAD resolution was achieved in all patients regardless of thawing time (100% vs 100%, p<0.001). On the other hand, the cumulative recurrence rate was significantly lower in 4HTG compared to 12HTG. (0% vs 30%, p=0.045). The observed species, Chao1, and Shannon indices for alpha-diversity measures revealed that there was greater microbial richness among groups in the post FMT group than in the pre FMT group. (p<0.001) A comparison of gut microbiota composition was performed using PCoA based on beta diversity of unweighted UniFrac distances. The 4HT group was found to be more similar to the donor than the 12HT group.

Conclusion

This study demonstrated that thawing time did not affect CDAD resolution but did make a difference in recurrence rate. This is the first study to show that the thawing time of frozen stools affects the CDAD recurrence rate and that 4-hour room temperature thawing should be preferred.

Introduction

Antidepressants are an integral part of the treatment of IBS as a disorder of gut-brain interaction (DGBI). Accordingly also a direct interaction with the gut microbiota might be relevant as an additional mode of action. With its high content of flavonoids, a substance group for which beneficial neuromodulatory effects, mediated by the microbiota, are known. St. John's wort extract, an antidepressant with clinically proven therapeutic equivalence to TCAs and SSRIs, could be a promising candidate for such an additional action, also as there is a history of use also in GI complaints.

Aims & Methods

Aim was to follow the test substance, a St. John's wort extract (STW 3-VI), with clinically proven anti-depressant activity [1] and standardized flavonoid content [2], through the GI tract. According to the Infogest protocol [3,4], first the influence of the upper GI digestion on the extract was modelled by subsequent incubaton with respective enzymes and buffer mixes. The predigested extract was subsequently incubated with fecal samples from ten healthy donors (24h, anoxic, 37°C). The metabolism of the flavonoids by the gut microbiota was captured by UHPLC-HRMS, and also proteolytic markers (BCFA and ammonium) were measured by GC-FID and IPB colorimetric analysis. 16S rRNA gene sequencing was used to assess shifts in microbial community composition, followed by functional analysis via ATLAS, using KEGG, Cazy and Pfam for annotations.

Results

All major extract constituents were stable towards upper intestinal tract digestion. They were, however, readily catabolized by the gut microbiota. Metabolites of the flavonoids included taxifolin, hydroxyphenylpropionic acids and protocatechuic acid. Proteolytic markers were significantly decreased. Differential abundance analysis of the microbial communities showed that several taxa were significantly modulated by the extract, with e.g. Bacteroides (p=0.005) Marinifilaceae (p=0.0007) and Prevotellaceae (p=0.035) decreasing. Functional analysis revealed 540 differently expressed genes (p<0.01), some involved in flavonoid metabolism.

Conclusion

Beyond the known antidepressant activity of St. John's wort extract, its components are also prodrugs for substances with potential therapeutic benefits in IBS as well as in psychic depression [5]. This is in accordance to the results of the functional analysis of the gut microbiota, given that changes of abundance of bacterial taxa by the treatment antagonize abundance shifts described in depressive as well as IBS patients [6]. The decrease of proteolytic markers is also in line with a beneficial action in IBS [7]. In conclusion, St. John's wort might, beyond its known efficacy in mild to moderate depression, also be a promising therapeutic option in IBS patients, especial with co-occurring depressive symptoms.

References

1. Gastpar M, Singer A, Zeller K. Efficacy and tolerability of hypericum extract STW3 in long-term treatment with a once-daily dosage in comparison with sertraline. Pharmacopsychiatry. 2005;38:78-86.
2. www.ema.europa.eu/en/medicines/herbal/hyperici-herba
3. Brodkorb A, Egger L, Alminger M, et al. INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat Protoc 2019; 14: 991-1014.
4. Pérez-Burillo S, Molino S, Navajas-Porras B. et al. An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality. Nat Protoc 2021; 16: 3186–3209.
5. Vissiennon C, Nieber K, Kelber O, Butterweck V. Route of administration determines the anxiolytic activity of the flavonols kaempferol, quercetin and myricetin -- are they prodrugs? J Nutr Biochem. 2012; 23: 733-740
6. Duan R, Zhu S, Wang B, Duan L. Alterations of gut microbiota in patients with irritable bowel syndrome based on 16S rRNA-targeted sequencing: A Systematic Review. Clin Transl Gastroenterol. 2019; 10:e00012.
7. Hou JJ, Wang X, Li Y, Su S, Wang YM, Wang BM. The relationship between gut microbiota and proteolytic activity in irritable bowel syndrome. Microb Pathog. 2021; 157:104995.

Disclosure

MEG, EMPF and RB have received fees from Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany, HEK and OK are employees of the same company.

Introduction

^​​​​​​T cells, are a major regulator and source of pro-inflammatory cytokines, which may promote colitis, but be protective in some cancers^1,2. Nevertheless, limited efforts have been made to explore factors which determine the phenotype of T cells and their impact on the above-mentioned phenomena. The adhesion G protein-coupled receptor F5 (ADGRF5) belongs to seven-transmembrane receptor subfamily and may be related to development of some cancer types³.

Aims & Methods

The aim of the study was to assess the significance of ADGRF5 for T cell function and its impact on colitis and colon cancer progression. WT and ADGRF5 knockout CD4⁺ T cells were examined for cytokine genes and proteins using real-time PCR, Western blot and multiplex analysis. Tumor killing assay and flow cytometry analysis were performed to evaluate tumor killing properties of T cells co-cultured with MC38 colon cancer cells. ADGRF5 WT and KO mice were utilized to examine the impact of ADGRF5 on colitis, colitis-associated cancer development and colon cancer growth. Adoptive transfer of WT and ADGRF5 knockout CD4⁺ and CD8⁺ T cells were examined in tumor models to assess their role in disease progression.

Results

In murine model of colon cancer, animals with ADGRF5 knockout developed significantly smaller tumors which was accompanied by enhanced CD3 expression and hyperproduction of cytokines related to T cell action compared to wild type mice. Ex vivo analysis of two main population of T cells obtained from spleen of wild type and ADGRF5 knockout mice revealed that activated CD4⁺ but not CD8⁺ T cells with ADGRF5 were manifested by increased production of major anti-cancer molecules such as interferon γ (IFN- γ), perforin and granzyme B, and were characterized by secretion profile of cytokines related to mixed phenotype of T helper (T_h) 1 and T_h2 cells. Moreover, we documented higher proportion of apoptotic cells in co-culture of MC38 colon cancer cells with ADGRF5 knockout but not wild type CD4⁺ T cells. Finally, in the murine model of colon cancer adoptive transfer of CD4⁺ T cells with ADGRF5 knockout led to a significant reduction of tumor volume which was reversed by administration of antibody against IFN-γ. Nevertheless, ADGRF5 knockout in mice with colitis-associated colorectal cancer and colitis was related to significantly higher number of colon tumors and worse disease activity index compared to wild type animals, respectively. These results together with the analysis of gene expression signature in inflamed colons suggest opposite role for ADGRF5 in colitis than in the allograft colon cancer.

Conclusion

ADGRF5 is a critical regulator of T cell function and specific knockout of ADGRF5 reprograms CD4⁺ T cells into the pro-inflammatory phenotype. Our in vitro and in vivo studies indicated that ADGRF5 knockout in T cells promote pro-inflammatory cytokines, which prevent gastrointestinal cancer progression while boost both colitis and colitis-associated colorectal cancer development.

References

1. Jacenik D., Karagiannidis I., Beswick E.J. 2022. T_h2 cells inhibit growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils. Br J Cancer. 128: 387-397. doi: 10.1038/s41416-022-02056-2.
2. Shale M., Schiering C., Powrie F. 2013. CD4⁺ T cell subsets in intestinal inflammation. Immunol Rev. 252: 164-82. doi: 10.1111/imr.12039.
3. Jacenik D., Hikisz P., Beswick E.J., Fichna J. 2023. The clinical relevance of the adhesion G protein-coupled receptor F5 for human diseases and cancers. Biochim Biophys Acta Mol Basis Dis. 1869: 166683 doi: 10.1016/j.bbadis.2023.166683.

Disclosure

The work was supported by the National Science Centre, Poland under research project no 2019/32/C/NZ5/00064 (DJ).