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Background and Aims: Hypercholesterolemia is frequent in people with primary biliary cholangitis (PBC); however, it does not seem to confer an increased risk of cardiovascular disease. We aimed to evaluate the prevalence of peripheral arterial disease in PBC women and its association with the gut–liver axis and systemic inflammation. Methods: Thirty patients affected by PBC and hypercholesterolemia were enrolled, with equal‐sized groups of women with non‐alcoholic fatty liver disease (NAFLD) and healthy controls (CTRL). All patients underwent Doppler ultrasound examination of peripheral arteries, assessment of flow‐mediated dilation, quantification of circulating cytokines and vasoactive mediators and characterization of the gut microbiota. Results: PBC patients had a higher prevalence of lower extremity arterial disease (LEAD) defined as atherosclerotic plaques in any of femoral, popliteal and/or tibial arteries compared with both NAFLD and CTRL women (83.3% vs. 53.3% and 50%, respectively; p =.01). Factors associated with LEAD at univariate analysis were VCAM‐1 (p =.002), ICAM‐1 (p =.003), and TNF‐alpha (p =.04) serum levels, but only VCAM‐1 (OR 1.1, 95% CI 1.0–1.1; p =.04) and TNF‐alpha (OR 1.12, 95% CI 0.99–1.26; p =.04) were confirmed as independent predictors in the multivariate model. Gut microbiota analysis revealed that Acidaminococcus (FDR = 0.0008), Bifidobacterium (FDR = 0.001) and Oscillospira (FDR = 0.03) were differentially expressed among groups. Acidaminococcus, which was increased in PBC, was positively correlated with TNF‐alpha serum levels. Down‐regulation of metabolic pathways linked to fatty acid and butyrate metabolism, glyoxylate metabolism and branched‐chain amino acids degradation was found in the functional gut metagenome of PBC women. Conclusions: LEAD is common in patients affected by PBC and is associated with inflammatory markers and alterations in the gut–liver axis. [ABSTRACT FROM AUTHOR]

Few data exist on differences in gut microbiota composition among principal gastrointestinal (GI) diseases. We evaluated the differences in gut microbiota composition among uncomplicated diverticular disease (DD), irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) patients. DD, IBS, and IBD patients along with healthy controls (CT) were enrolled in our Italian GI outpatient clinic. Stool samples were collected. Microbiota composition was evaluated through a metagenomic gene-targeted approach. GI pathology represented a continuous spectrum of diseases where IBD displayed one extreme, while CT displayed the other. Among Phyla, Biplot PC2/PC3 and dendogram plot showed major differences in samples from IBS and IBD. DD resembled species CT composition, but not for Bacteroides fragilis. In IBS, Dialister spp. and then Faecalibacterium prausnitzii were the most representative species. Ulcerative colitis showed a reduced concentration of Clostridium difficile and an increase of Bacteroides fragilis. In Crohn's disease, Parabacteroides distasonis was the most represented, while Faecalibacterium prausnitzii and Bacteroides fragilis were significantly reduced. Each disorder has its definite overall microbial signature, which produces a clear differentiation from the others. On the other hand, shared alterations constitute the “core dysbiosis” of GI diseases. The assessment of these microbial markers represents a parameter that may complete the diagnostic assessment. [ABSTRACT FROM AUTHOR]

Antibiotics are mainly used in clinical practice for their activity against pathogens, but they also alter the composition of commensal gut microbial community. Rifaximin is a non-absorbable antibiotic with additional effects on the gut microbiota about which very little is known. It is still not clear to what extent rifaximin can be able to modulate gut microbiota composition and diversity in different clinical settings. Studies based on culture-dependent techniques revealed that rifaximin treatment promotes the growth of beneficial bacteria, such as Bifidobacteria and Lactobacilli. Accordingly, our metagenomic analysis carried out on patients with different gastrointestinal and liver diseases highlighted a significant increase in Lactobacilli after rifaximin treatment, persisting in the short time period. This result was independent of the disease background and was not accompanied by a significant alteration of the overall gut microbial ecology. This suggests that rifaximin can exert important eubiotic effects independently of the original disease, producing a favorable gut microbiota perturbation without changing its overall composition and diversity. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) is a microorganism with a pronounced capability of adaptation under environmental stress solicitations. Its persistence and antimicrobial resistance to the drugs commonly used in the anti-H. pylori therapy are associated with the development of a biofilm mainly composed of DNA, proteins, and polysaccharides. A fundamental step to increase the success of clinical treatments is the development of new strategies and molecules able to interfere with the biofilm architecture and thus able to enhance the effects of antibiotics. By using Atomic Force Microscopy and Scanning Electron Microscopy we analyzed the effects of the alginate lyase (AlgL), an enzyme able to degrade a wide class of polysaccharides, on the H. pylori shape, surface morphology, and biofilm adhesion properties. We demonstrated that AlgL generates a noticeable loss of H. pylori coccoid form in favor of the bacillary form and reduces the H. pylori extracellular polymeric substances (EPS). [ABSTRACT FROM AUTHOR]

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine class of toxins secreted from certain fungi including Aspergillus fumigatus, which is the most prolific producer of this secondary metabolite. Recently, enhanced amounts of GT were found in in vitro biofilm-grown A. fumigatus mycelium. To further correlate the A. fumigatus biofilm growth phenotype with the enhanced secretion of GT, a polyclonal antibody (pAb) was produced by immunizing mice against GT. By an indirect immunofluorescent assay, pAb was then able to recognize specifically GT onto A. fumigatus Af293 biofilm formed on human pulmonary epithelial cells. Then, treating Af293 biofilms with a compound which reduces the GT disulfide bonds provoked shutdown of the GT-specific immunofluorescence ( IF) signals along the hyphae. To explore the potential of GT for diagnostic use, pAb was shown to react with GT on hyphae into Aspergillus culture-positive respiratory tract specimens from patients with probable invasive aspergillosis ( IA) and into tissue specimens from the lungs of patients with proven IA. As the presence of fungal hyphae in clinical specimens strongly indicates the in vivo A. fumigatus growth as a biofilm, anti-GT antibodies could be a specific and sensitive diagnostic tool for detecting A. fumigatus biofilm-associated clinical infections. [ABSTRACT FROM AUTHOR]

Background & Aim: Sarcopenia is frequent in cirrhosis and is associated with unfavourable outcomes. The role of the gut-liver-muscle axis in this setting has been poorly investigated. The aim of this study was to identify gut microbiota, metabolic and inflammatory signatures associated with sarcopenia in cirrhotic patients., Methods: Fifty cirrhotic patients assessed for the presence of sarcopenia by the quantification of muscle mass and strength were compared with age- and sex-matched controls. A multiomic analysis, including gut microbiota composition and metabolomics, serum myokines and systemic and intestinal inflammatory mediators, was performed., Results: The gut microbiota of sarcopenic cirrhotic patients was poor in bacteria associated with physical function (Methanobrevibacter, Prevotella and Akkermansia), and was enriched in Eggerthella, a gut microbial marker of frailty. The abundance of potentially pathogenic bacteria, such as Klebsiella, was also increased, to the detriment of autochthonous ones. Sarcopenia was associated with elevated serum levels of pro-inflammatory mediators and of fibroblast growth factor 21 (FGF21) in cirrhotic patients. Gut microbiota metabolic pathways involved in amino acid, protein and branched-chain amino acid metabolism were up-regulated, in addition to ethanol, trimethylamine and dimethylamine production. Correlation networks and clusters of variables associated with sarcopenia were identified, including one centred on Klebsiella/ethanol/FGF21/Eggerthella/Prevotella., Conclusions: Alterations in the gut-liver-muscle axis are associated with sarcopenia in patients with cirrhosis. Detrimental but also compensatory functions are involved in this complex network., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Systemic sclerosis (SSc) is a rare multi-organ disorder with a prominent gastrointestinal (GI) involvement. Altered gut microbiota is now considered a pivotal factor associated with the development of immune-mediated and inflammatory diseases. We performed a 16S ribosomal RNA (rRNA) gene-sequencing analysis of fecal microbiota in a cohort of SSc patients and matched healthy controls (HCs), with the aim to obtain some hints about a possible role of dysbiosis in the onset, progression, and severity of the disease., Methods: We analysed stool samples from 63 SSc patients with different disease duration, phenotype, and nutritional status and from 17 HCs through 16S ribosomal RNA (rRNA) gene-sequencing., Results: Microbial richness was lower for patients with long-standing disease. A similar observation was made for patients with diffuse cutaneous SSc (dsSSc) compared to those with limited variant (lcSSc) and for patients who reported a recent weight loss. Consistent with previous reports, we noted a deviation of the intestinal microbial composition in patients with SSc compared to HCs, with a greater expression of Lactobacillus and Streptococcus and a depletion of Sutterella. Nutritional status, assessed using BMI as a surrogate, appeared to have a marked impact on the gut microbiota, with overweight patients showing lower richness compared both to underweight and normal-BMI patients., Conclusions: Our findings expand the current knowledge of gut microbiota in SSc and could be useful to identify patients who would most benefit from treatments aimed at restoring the eu-biosis.

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 glycoprotein (HCV/E2) represents a major target antigen involved in the containment and resolution of naturally occurring HCV infection. Antibody phage display allows the molecular cloning of cDNA sequences encoding antibody fragments specific to a wide range of diverse antigens. These antibodies may be produced in bacteria as Fab or converted into full length IgG. The latter have a higher serum half life and display Fc encoded function. Using a library prepared from an HCV-infected individual, we selected a panel of Fab fragments for binding to invariant epitopes of the E2 glycoprotein. This work describes a technique used to convert the selected Fab fragments into full length IgG and to express these antibodies in eukaryotic cells. All the recombinant antibodies retained the binding specificity of the parental Fab showing an increase in apparent relative affinity for E2.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.