Bacteroides uniformis CECT 7771 requires adaptive immunity to improve glucose tolerance but not to prevent body weight gain in diet-induced obese mice.

BACKGROUND: The metabolic disturbances of obesity can be mitigated by strategies modulating the gut microbiota. In this study, we sought to identify whether innate or adaptive immunity mediates the beneficial metabolic effects of the human intestinal bacterium Bacteroides uniformis CECT 7771 in obesity. METHODS: We evaluated the effects of orally administered B. uniformis on energy homeostasis, intestinal immunity, hormone levels, and gut microbiota in wild-type and Rag1-deficient mice with diet-induced obesity. We also assessed whether B. uniformis needed to be viable to exert its beneficial effects in obesity and to directly induce immunoregulatory effects. RESULTS: The administration of B. uniformis to obese mice improved glucose tolerance and insulin secretion, restored the caloric intake suppression after an oral glucose challenge, and reduced hyperglycemia. The pre- and post-prandial glucose-related benefits were associated with restoration of the anti-inflammatory tone mediated by type 2 macrophages and regulatory T cells (Tregs) in the lamina propria of the small intestine. Contrastingly, B. uniformis administration failed to improve glucose tolerance in obese Rag1-/- mice, but prevented the increased body weight gain and adiposity. Overall, the beneficial effects seemed to be independent of enteroendocrine effects and of major changes in gut microbiota composition. B. uniformis directly induced Tregs generation from naïve CD4+ T cells in vitro and was not required to be viable to improve glucose homeostasis but its viability was necessary to prevent body weight gain in diet-induced obese wild-type mice. CONCLUSIONS: Here we demonstrate that B. uniformis modulates the energy homeostasis in diet-induced obese mice through different mechanisms. The bacterium improves oral glucose tolerance by adaptive immunity-dependent mechanisms that do not require cell viability and prevents body weight gain by adaptive immunity-independent mechanisms which require cell viability. Video Abstract.

Development of Ciprofloxacin-Loaded Electrospun Yarns of Application Interest as Antimicrobial Surgical Suture Materials.

Surgical site infections (SSI) occur very frequently during post-operative procedures and are often treated with oral antibiotics, which may cause some side effects. This type of infection could be avoided by encapsulating antimicrobial/anti-inflammatory drugs within the surgical suture materials so that they can more efficiently act on the site of action during wound closure, avoiding post-operative bacterial infection and spreading. This work was aimed at developing novel electrospun bio-based anti-infective fibre-based yarns as novel suture materials for preventing surgical site infections. For this, yarns based on flying intertwined microfibres (1.95 ± 0.22 µm) were fabricated in situ during the electrospinning process using a specially designed yarn collector. The electrospun yarn sutures (diameter 300-500 µm) were made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with different contents of 3HV units and contained ciprofloxacin hydrochloride (CPX) as the antimicrobial active pharmaceutical ingredient (API). The yarns were then analysed by scanning electron microscopy, Fourier transform infrared spectroscopy, wide-angle X-ray scattering, differential scanning calorimetry, and in vitro drug release. The yarns were also analysed in terms of antimicrobial and mechanical properties. The material characterization indicated that the varying polymer molecular architecture affected the attained polymer crystallinity, which was correlated with the different drug-eluting profiles. Moreover, the materials exhibited the inherent stiff behaviour of PHBV, which was further enhanced by the API. Lastly, all the yarn sutures presented antimicrobial properties for a time release of 5 days against both Gram-positive and Gram-negative pathogenic bacteria. The results highlight the potential of the developed antimicrobial electrospun yarns in this study as potential innovative suture materials to prevent surgical infections.

Nanostructured Valsartan Microparticles with Enhanced Bioavailability Produced by High-Throughput Electrohydrodynamic Room-Temperature Atomization.

The high-throughput drying and encapsulation technique called electrospraying assisted by pressurized gas (EAPG) was used for the first time to produce nanostructured valsartan within microparticles of excipients. Valsartan, a poorly absorbed and lipid-soluble drug, was selected since it is considered a good model for BCS class II drugs. Two different polymeric matrices were selected as excipients, i.e., hydroxypropyl methylcellulose (HPMC) and lactose monohydrate, while Span 20 was used as a surfactant. The produced 80% valsartan loading formulations were characterized in terms of morphology, crystallinity, in vitro release, in vitro Caco-2 cells' permeability, and in vivo pharmacokinetic study. Spherical microparticles of ca. 4 µm were obtained within which valsartan nanoparticles were seen to range from 150 to 650 nm. Wide-angle X-ray scattering and differential scanning calorimetry confirmed that valsartan had a lower and/or more ill-defined crystallinity than the commercial source, and photon correlation spectroscopy and transmission electron microscopy proved that it was dispersed and distributed in the form of nanoparticles of controlled size. In vitro dissolution tests showed that the HPMC formulation with the lowest API particle size, i.e., 150 nm, dissolved 2.5-fold faster than the commercial valsartan in the first 10 min. This formulation also showed a 4-fold faster in vitro permeability than the commercial valsartan and a 3-fold higher systemic exposure than the commercial sample. The results proved the potential of the EAPG processing technique for the production of safe-to-handle microparticles containing high quantities of a highly dispersed and distributed nanonized BCS class II model drug with enhanced bioavailability.

Interlaboratory Comparative Study to Detect Potentially Infectious Human Enteric Viruses in Influent and Effluent Waters.

Wastewater represents the main reusable water source after being adequately sanitized by wastewater treatment plants (WWTPs). In this sense, only bacterial quality indicators are usually checked to this end, and human pathogenic viruses usually escape from both sanitization procedures and controls, posing a health risk on the use of effluent waters. In this study, we evaluated a protocol based on aluminum adsorption-precipitation to concentrate several human enteric viruses, including norovirus genogroup I (NoV GI), NoV GII, hepatitis A virus (HAV), astrovirus (HAstV), and rotavirus (RV), with limits of detection of 4.08, 4.64, 5.46 log genomic copies (gc)/L, 3.31, and 5.41 log PCR units (PCRU)/L, respectively. Furthermore, the method was applied in two independent laboratories to monitor the presence of NoV GI, NoV GII, and HAV in effluent and influent waters collected from five WWTPs at two different sampling dates. Concomitantly, a viability PMAxx-RT-qPCR was applied to all the samples to get information on the potential infectivity of both influent and effluent waters. The ranges of the titers in influent waters for NoV GI, NoV GII, RV, and HAstV were 4.80-7.56, 5.19-7.31 log gc/L, 5.41-6.52, and 4.59-7.33 log PCRU/L, respectively. In effluent waters, the titers ranged between 4.08 and 6.27, 4.64 and 6.08 log gc/L, < 5.51, and between 3.31 and 5.58 log PCRU/L. Moreover, the viral titers detected by viability RT-qPCR showed statistical differences with RT-qPCR alone, suggesting the potential viral infectivity of the samples despite some observed reductions. The proposed method could be applied in ill-equipped laboratories, due to the lack of a requirement for a specific apparatus (i.e., ultracentrifuge).

VSL#3 probiotic differently influences IEC-6 intestinal epithelial cell status and function.

The data here reported introduce the wound-healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi-strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non-transformed small-intestinal epithelial cell line, IEC-6. The effects on cellular morphology, viability, migration, and H2 O2 -induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA-sourced product ("USA-made") VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy-derived product ("Italy-made") VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with "USA-made" lysate led to a higher rate of wounded monolayer healing, while the addition of "Italy-made" bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from "USA-made" VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, "Italy-made" lysates had not similar effect. "USA-made" lysate was able to cause a total inhibition of H2 O2 -induced cytotoxic effect whereas "Italy-made" VSL#3 lysate was unable to protect IEC-6 cells from H2 O2 -induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of "USA-made" VSL#3 lysates, as well as the idea that "Italy-made" formulation was unable to prevent significantly the H2 O2 -induced oxidative stress.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process.

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-ß antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc.

Immunophenotypic characterization of human glioblastoma stem cells: correlation with clinical outcome.

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. ß-tubulin III, nestin, CD133, GFAP, and SOX-2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX-2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression-free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX-2 may be useful to identify patients at risk of disease progression.