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Eosinophils were previously reported to play a role in intestinal inflammation and fibrosis. Whether this is as a bystander or as an active participant is still up for debate. Moreover, data describing a causal relationship between eosinophils and intestinal fibrosis are scarce. We here aimed to elucidate the role of eosinophils in the pathogenesis of intestinal inflammation and fibrosis. Therefore, we stimulated fibroblasts with (active) eosinophils or with Eosinophil Cationic Protein (ECP), and assessed fibroblast activation via flow cytometry and immunocytochemistry. We observed decreased fibroblast activation when fibroblasts were co-cultured with active eosinophils or after stimulation with ECP in comparison to monoculture conditions, but not in case of co-culturing with inactivated eosinophils. Furthermore, eosinophil depletion in a RAG -/- chronic DSS colitis model resulted in decreased inflammation, but increased development of fibrosis. In this model, we could show increased expression of the anti-inflammatory protein IL-10 and the pro-fibrotic factors IL-1β, FGF-21 and TGF-β3 in the eosinophil-depleted mice compared to the control mice. In conclusion, our in vitro data revealed an anti-fibrotic role for eosinophils. In line, in a chronic murine colitis model, we observed a pro-inflammatory, but an anti-fibrotic, role for eosinophils. Furthermore, we identified an increased presence of anti-inflammatory and pro-fibrotic cytokines in the eosinophil depleted group., (© 2024. The Author(s).)

Acetate-producing Saccharomyces cerevisiae var. boulardii strains could exert improved effects on ulcerative colitis, which here, was preclinically evaluated in an acute dextran sodium sulphate induced model of colitis. Nine-week-old female mice were divided into 12 groups, receiving either drinking water or 2.75% dextran sodium sulphate for 7 days, combined with a daily gavage of various treatments with different levels of acetate accumulation: sham control (phosphate buffered saline, no acetate), non-probiotic control (Baker's yeast, no acetate), probiotic control (Enterol ® , transient acetate), and additionally several Saccharomyces cerevisiae var. boulardii strains with respectively no, high, and extra-high acetate accumulation. Disease activity was monitored daily, and feces samples were collected at different timepoints. On day 14, the mice were sacrificed, upon which blood and colonic tissue were collected for analysis. Disease activity in inflamed mice was lower when treated with the high-acetate-producing strain compared to sham and non-probiotic controls. The non-acetate-producing strain showed higher disease activity compared to the acetate-producing strains. Accordingly, higher histologic inflammation was observed in non- or transient-acetate-producing strains compared to the sham control, whereas this increase was not observed for high- and extra-high-acetate-producing strains upon induction of inflammation. These anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes. Moreover, only the strain with the highest acetate production was superior in maintaining a stable gut microbial alpha-diversity upon inflammation. These findings support new possibilities for acetate-mediated management of inflammation in inflammatory bowel disease by administrating high-acetate-producing Saccharomyces cerevisae var. boulardii strains.

Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2 S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2 S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4 . It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii .IMPORTANCESuperior variants of the probiotic yeast Saccharomyces boulardii produce high levels of acetic acid, which inhibit the growth of bacterial pathogens. However, these strains also show increased acid sensitivity, which can compromise the viability of the cells during their passage through the stomach. In this work, we have developed by genetic engineering a variant of Saccharomyces boulardii that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the S. boulardii yeasts with higher acetic acid production persist longer in the gut, in agreement with a previous work indicating competition between probiotic yeast and bacteria for residence in the gut., Competing Interests: B.T.D.C., P.V., and J.M.T. are inventors on a patent application related to this work (EP 22153700.4.). J.M.T. is the owner/managing director of NovelYeast bv with commercial interest in engineered Saccharomyces boulardii.

Background & Aims: Rigorous donor preselection on microbiota level, strict anaerobic processing, and repeated fecal microbiota transplantation (FMT) administration were hypothesized to improve FMT induction of remission in ulcerative colitis (UC)., Methods: The RESTORE-UC trial was a multi-centric, double-blind, sham-controlled, randomized trial. Patients with moderate to severe UC (defined by total Mayo 4-10) were randomly allocated to receive 4 anaerobic-prepared allogenic or autologous donor FMTs. Allogenic donor material was selected after a rigorous screening based on microbial cell count, enterotype, and the abundance of specific genera. The primary endpoint was steroid-free clinical remission (total Mayo ≤2, no sub-score >1) at week 8. A pre-planned futility analysis was performed after 66% (n = 72) of intended inclusions (n = 108). Quantitative microbiome profiling (n = 44) was performed at weeks 0 and 8., Results: In total, 72 patients were included, of which 66 received at least 1 FMT (allogenic FMT, n = 30 and autologous FMT, n = 36). At week 8, respectively, 3 and 5 patients reached the primary endpoint of steroid-free clinical remission (P = .72), indicating no treatment difference of at least 5% in favor of allogenic FMT. Hence, the study was stopped due to futility. Microbiome analysis showed numerically more enterotype transitions upon allogenic FMT compared with autologous FMT, and more transitions were observed when patients were treated with a different enterotype than their own at baseline (P = .01). Primary response was associated with lower total Mayo scores, lower bacterial cell counts, and higher Bacteroides 2 prevalence at baseline., Conclusion: The RESTORE-UC trial did not meet its primary endpoint of increased steroid-free clinical remission at week 8. Further research should additionally consider patient selection, sterilized sham-control, increased frequency, density, and viability of FMT prior to administration., Clinicaltrials: gov, Number: NCT03110289., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers ( n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

The gut microbiome and the intestinal immune system are driving contributors to inflammatory bowel diseases (IBD). Both have an important signalling factor in common: short-chain fatty acids (SCFAs). SCFAs (acetate, propionate and butyrate) are produced by bacterial fermentation in the gut and exert several effects on host metabolism and immune system. This review provides an overview of the current knowledge of these effects, with specific focus on energy metabolism, intestinal barrier, immune system, and disease activity in IBD. To conclude, more research is needed on the cross-feeding mechanisms in the gut microbiome, as well as on the therapeutic potential of SCFAs on different disease models. Also randomized controlled trials and prospective cohort studies should investigate the clinical impact of SCFA administration., Competing Interests: Declaration of Competing Interest KM reports employment as a Pfizer Medical advisor in inflammation and immunology since 5/01/2021. JR reports grants from J&J, Beneo, Colruyt, Prodigest, Aphea.Bio, Cargill, other from Yakult, Nutricia, GSK Vaccines, MRM health, Aphea.Bio, Janssen Pharmaceuticals, Roche, Takeda, Tsumura, ABInbev, DSM, Nestle, Pfizer, BMS,  Ferring, outside the submitted work; KV reports grants from Nestlé, personal fees from Yakult, personal fees from Biocodex, outside the submitted work; SV reports grants from AbbVie, J&J, Pfizer, and Takeda, other from AbbVie, Arena Pharmaceuticals, Avaxia, Boehringer Ingelheim, Celgene, Dr Falk Pharma, Ferring, Galapagos, Genentech-Roche, Gilead, Hospira, Janssen, Mundipharma, MSD, Pfizer, Prodigest, Progenity, Prometheus, Robarts Clinical Trials, Second Genome, Shire, Takeda, Theravance, and Tillots Pharma AG,  outside the submitted work; The remaining authors disclose no conflicts., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: The aim of this study was to evaluate the drug-drug interaction between pimodivir, a novel, non-nucleoside polymerase basic protein 2 (PB2) subunit inhibitor of the influenza A virus polymerase complex, and oseltamivir, to assess the feasibility of this combination therapy. Furthermore, single- and multiple-dose pharmacokinetics and safety of pimodivir in healthy volunteers were assessed., Methods: In Part 1 of this open-label Phase 1 study, healthy volunteers (n = 18) were randomized to one of six cross-over treatment sequences, each comprising administration of oseltamivir 75 mg or pimodivir 600 mg or combination thereof twice daily on Days 1-4, followed by a single morning dose on Day 5. Between each treatment session, there was a minimum 5-day washout period. In Part 2, healthy volunteers (n = 16) randomly received pimodivir 600 mg or placebo (3:1) twice daily on Days 1-9, followed by a single morning dose on Day 10. Pharmacokinetics of pimodivir, oseltamivir and oseltamivir carboxylate, and safety were assessed., Results: In Part 1, co-administration of pimodivir with oseltamivir increased the C max of pimodivir by 31% (90% CI: 0.92-1.85) with no change in C min or AUC 12h . Pimodivir had no effect on oseltamivir or oseltamivir carboxylate pharmacokinetics. In Part 2, after single- and multiple-dose administration of pimodivir, there was a 1.2- and 1.8-fold increase in C max and AUC 12h , respectively, between Day 1 and Day 10. The most frequently reported treatment-emergent adverse event was diarrhoea (n = 7 each in Part 1 and 2)., Conclusion: Combination treatment with pimodivir and oseltamivir in healthy volunteers showed no clinically relevant drug-drug interactions. No safety concerns were identified with pimodivir 600 mg twice daily alone or in combination with oseltamivir 75 mg twice daily., (© 2018 The British Pharmacological Society.)

Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

Studies [Zhou, D., Chen, L.-M., Hernandez, L., Shears, S.B., and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

The stimulation of thyroid cell proliferation by TSH through cAMP depends on permissive comitogenic factors, generally the insulin-like growth factors and insulin. In dog thyroid primary cultures, the use of the phosphodiesterase-resistant analog of cAMP (Bu)(2)cAMP instead of TSH allowed to unveil a potent comitogenic activity of carbamylcholine, which can substitute for insulin and was shown to mimic insulin action on cell cycle regulatory proteins. Like insulin, carbamylcholine induced the accumulation of cyclin D3 and overcame the repression by cAMP of this protein, which was shown 1) to be essential for cell cycle progression by means of microinjections of a neutralizing antibody; and 2) to be rate limiting for the cAMP-dependent assembly of cyclin D3-cdk4 complexes, their nuclear translocation and the phosphorylation of pRb. Relative to insulin, carbamylcholine offers the significant experimental advantage that its signaling cascades can be immediately deactivated by the muscarinic antagonist atropine. In the presence of carbamylcholine, the elimination of (Bu)(2)cAMP blocked within 2 h the entry of cells into DNA synthesis phase, but the addition of atropine still permitted the entry of cells in S phase. These data support our view that the progression in G1 phase stimulated by cAMP consists of at least two essential actions that are clearly dissociated: in a first stage, depending on the supportive activity of an agent that stimulates the required cyclin D3 accumulation, cAMP induces the assembly and nuclear translocation of cyclin D3-cdk4 complexes, and then cAMP can exert alone the last crucial control that determines the cell commitment toward DNA replication.