Lacticaseibacillus rhamnosus GG Counteracts Rotavirus-Induced Ion Secretion and Enterocyte Damage by Inhibiting Oxidative Stress and Apoptosis Through Specific Effects of Living and Postbiotic Preparations

Background: Administration of Lacticaseibacillus rhamnosus GG (LGG) to children with gastroenteritis is recommended by universal guidelines. Rotavirus (RV) causes diarrhea through combined cytotoxic and enterotoxic effects. Aim of this study was to evaluate the mechanisms of efficacy of LGG in an in-vitro model of RV diarrhea in its viable form (LGG) and conditioned medium (mLGG). Methods: Ion secretion corresponding to the NSP4 enterotoxic effect, was evaluated by short circuit current (Isc) and the cytotoxic effect by transepithelial electrical resistance (TEER) in Ussing chambers, upon exposure to RV in Caco-2 enterocyte monolayers treated or not with living probiotic or its culture supernatant. Mechanisms of enterotoxic and cytotoxic damage were evaluated including oxidative stress measured by reactive oxygen species, apoptosis evaluated by DAPI and nuclear staining, NFkβ immunofluorescence. Results: RV induced Isc increase and TEER decrease, respectively indicating ion secretion and epithelial damage, the two established pathways of diarrhea. Both probiotic preparations reduced both diarrheal effects, but their potency was different. Live LGG was equally effective on both enterotoxic and cytotoxic effect whereas mLGG was highly effective on ion secretion and showed minimal protective effects on cytoskeleton, apoptosis and NFkβ. Conclusions: LGG counteracts RV-induced diarrhea by inhibiting both cytotoxic and enterotoxic pathogenic mechanisms. Namely, LGG inhibits chloride secretion by specific moieties secreted in the medium with a direct pharmacologic-like action. This is considered a postbiotic effect. Subsequently, live bacteria exert a probiotic effect protecting the enterocyte structure

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor

Loss of Guanylyl Cyclase C (GCC) Signaling Leads to Dysfunctional Intestinal Barrier

Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC-/-) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN-/-) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC-/- and UGN-/- mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC-/- mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC-/- and UGN-/- mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury

Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication

HIV-1 replication results in mitochondrial damage that is enhanced during antiretroviral therapy (ART). The onset of HIV-1 replication is regulated by viral protein Tat, a 101-residue protein codified by two exons that elongates viral transcripts. Although the first exon of Tat (aa 1–72) forms itself an active protein, the presence of the second exon (aa 73–101) results in a more competent transcriptional protein with additional functions. Results: Mitochondrial overall functions were analyzed in Jurkat cells stably expressing full-length Tat (Tat101) or one-exon Tat (Tat72). Representative results were confirmed in PBLs transiently expressing Tat101 and in HIV-infected Jurkat cells. The intracellular expression of Tat101 induced the deregulation of metabolism and cytoskeletal proteins which remodeled the function and distribution of mitochondria. Tat101 reduced the transcription of the mtDNA, resulting in low ATP production. The total amount of mitochondria increased likely to counteract their functional impairment. These effects were enhanced when Tat second exon was expressed. Conclusions: Intracellular Tat altered mtDNA transcription, mitochondrial content and distribution in CD4+ T cells. The importance of Tat second exon in non-transcriptional functions was confirmed. Tat101 may be responsible for mitochondrial dysfunctions found in HIV-1 infected patients.We greatly appreciate the secretarial assistance of Mrs Olga Palao. This work was supported by FIPSE (360924/10), Spanish Ministry of Economy and Competitiveness (SAF2010-18388), Spanish Ministry of Health (EC11- 285), AIDS Network ISCIII-RETIC (RD12/0017/0015), Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness (FIS PI12/00506). The work of Sara Rodríguez-Mora is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness (2013). The work of María Rosa López-Huertas is supported by a fellowship of the European Union Programme Health 2009 (CHAARM).S

The Role of Purported Mucoprotectants in Dealing with Irritable Bowel Syndrome, Functional Diarrhea, and Other Chronic Diarrheal Disorders in Adults

Chronic diarrhea is a frequent presenting symptom, both in primary care medicine and in specialized gastroenterology units. It is estimated that more than 5% of the global population suffers from chronic diarrhea. and that about 40% of these subjects are older than 60 years. The clinician is frequently faced with the need to decide which is the best therapeutic approach for these patients. While the origin of chronic diarrhea is diverse, impairment of intestinal barrier function, dysbiosis. and mucosal micro-inflammation are being increasingly recognized as underlying phenomena characterizing a variety of chronic diarrheal diseases. In addition to current pharmacological therapies, there is growing interest in alternative products such as mucoprotectants, which form a mucoadhesive film over the epithelium to reduce and protect against the development of altered intestinal permeability, dysbiosis, and mucosal micro-inflammation. This manuscript focuses on chronic diarrhea in adults, and we will review recent evidence on the ability of these natural compounds to improve symptoms associated with chronic diarrhea and to exert protective effects for the intestinal barrier

Acute Infectious Diarrhea

Acute infectious diarrhea (AID) is one of the most common diseases in pediatric age with relevant burden both in high- and in low-income countries.Thanks to their direct action on enterocyte functions and indirect actions on mucosal and systemic immune system and intestinal microenvironment, probiotics are an ideal intervention to manage AID in childhood. However, their efficacy is strictly related to strains and indications, and practitioners should take this information into account in clinical practice.This chapter summarizes the main mechanisms of action of probiotics in AID, with a focus on proof of efficacy supporting their use in prevention and treatment of infant AID.The use of selected strains in appropriate doses is strongly recommended by guidelines of AID, based on large and consistent proofs of efficacy and safety. At present, therapy with probiotics of AID is arguably the strongest indication for probiotics in medicine. Future research should investigate probiotic efficacy in at-risk populations and settings where the evidence is missing.Their role in prevention of AID is however questionable in healthy population, whereas it should be considered in at-risk population. Evidence for prevention of diarrhea in day-care centers and communities is lacking, but consistent evidence supports efficacy in prevention of hospital acquired diarrhea.Overall, AID is the most convincing area for probiotic use in children, and effective strains should be used early after onset of symptoms

Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration

AIMS: To assess the incidence of intestinal inflammation in children with cystic fibrosis and to investigate whether probiotics decrease it. STUDY DESIGN: In this two-phase, controlled, prospective study, faecal calprotectin was measured by enzyme-linked immunosorbent assay in 30 children with cystic fibrosis, 30 healthy controls and 15 children with active inflammatory bowel disease. Ten children with cystic fibrosis received Lactobacillus GG, and faecal calprotectin was re-measured 4 weeks later. Rectal nitric oxide production was measured with the rectal dialysis bag technique in 20 children with cystic fibrosis, 20 healthy controls and 15 children with inflammatory bowel disease. Five children with cystic fibrosis received Lactobacillus GG and nitric oxide was re-measured 4 weeks later. RESULTS: Mean faecal calprotectin was significantly higher in the two groups of patients than in controls. Abnormal values were detected in 27 of 30 cystic fibrosis and in 15 of 15 inflammatory bowel disease children. Also mean nitric oxide production was increased in both group of patients, and abnormal values were detected in 19 of 20 cystic fibrosis and in 15 of 15 inflammatory bowel disease children. Calprotectin and nitric oxide concentrations were reduced after probiotics administration. CONCLUSIONS: Intestinal inflammation is a major feature of cystic fibrosis and is reduced by probiotics. The latter finding suggests that intestinal microflora play a major role in intestinal inflammation in cystic fibrosis children