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In a previously published double-blind, placebo-controlled study, we showed that probiotics intake exerted a positive effect on sleep quality and a general improvement across time in different aspects of the profile of mood state, like sadness, anger, and fatigue in 33 healthy individuals. The present work investigates the impact of the probiotic product, constituted of Limosilactobacillus fermentum LF16, Lacticaseibacillus rhamnosus LR06, Lactiplantibacillus plantarum LP01 (all former members of Lactobacillus genus), and Bifidobacterium longum 04, on the gut microbiota composition of the same cohort through a metabarcoding analysis. Both the placebo and probiotic treatments had a significant impact on the microbiota composition. Statistical analysis showed that the microbiota of the individuals could be clustered into three groups, or bacteriotypes, at the baseline, and, inherently, bacterial compositions were linked to different responses to probiotic and placebo intakes. Interestingly, L. rhamnosus and L. fermentum were retrieved in the probiotic-treated cohort, while a bifidogenic effect of maltodextrin, used as placebo, was observed. The present study shed light on the importance of defining bacteriotypes to assess the impact of interventions on the gut microbiota and allowed to reveal microbial components which could be related to positive effects (i.e. sleep quality improvement) to be verified in further studies.

Background A connection between amyotrophic lateral sclerosis (ALS) and altered gut microbiota composition has previously been reported in animal models. This work is the first prospective longitudinal study addressing the microbiota composition in ALS patients and the impact of a probiotic supplementation on the gut microbiota and disease progression. Methods Fifty patients and 50 matched controls were enrolled. The microbial profile of stool samples from patients and controls was analyzed via PCR-Denaturing Gradient Gel Electrophoresis, and the main microbial groups quantified via qPCR. The whole microbiota was then analyzed via next generation sequencing after amplification of the V3–V4 region of 16S rDNA. Patients were then randomized to receive probiotic treatment or placebo and followed up for 6 months with ALSFRS-R, BMI, and FVC%. Results The results demonstrate that the gut microbiota of ALS patients is characterized by some differences with respect to controls, regardless of the disability degree. Moreover, the gut microbiota composition changes during the course of the disease as demonstrated by the significant decrease in the number of observed operational taxonomic unit during the follow-up. Interestingly, an unbalance between potentially protective microbial groups, such as Bacteroidetes, and other with potential neurotoxic or pro-inflammatory activity, such as Cyanobacteria, has been shown. The 6-month probiotic treatment influenced the gut microbial composition; however, it did not bring the biodiversity of intestinal microbiota of patients closer to that of control subjects and no influence on the progression of the disease measured by ALSFRS-R was demonstrated. Conclusions Our study poses the bases for larger clinical studies to characterize the microbiota changes as a novel ALS biomarker and to test new microbial strategy to ameliorate the health status of the gut. Trial registration CE 107/14, approved by the Ethics Committee of the “Maggiore della Carità” University Hospital, Italy.

Autism spectrum disorders (ASDs) represent a diagnostic challenge with a still partially uncertain etiology, in which genetic and environmental factors have now been assessed. Among the hypotheses underlying the involvement of biological and environmental factors, the gut–brain axis is of particular interest in autism spectrum disorders. Several studies have highlighted the related incidence of particular gastrointestinal symptoms (GISs) in children suffering from ASDs. Probiotics have shown success in treating several gastrointestinal dysbiotic disorders; therefore, it is plausible to investigate whether they can alleviate behavioral symptoms as well. On these bases, a randomized double-blind crossover study with a placebo was conducted, evaluating the effects of a mixture of probiotics in a group of 61 subjects aged between 24 months and 16 years old with a diagnosis of ASD. Behavioral evaluation was performed through the administration of a questionnaire including a Parenting Stress Index (PSI) test and the Vineland Adaptive Behavior Scale (VABS). The Psycho-Educational Profile and the Autism Spectrum Rating Scale (ASRS) were also evaluated. Microbial composition analyses of fecal samples of the two groups was also performed. The study showed significant improvements in GISs, communication skills, maladaptive behaviors, and perceived parental stress level after the administration of probiotics. Microbiome alpha diversity was comparable between treatment arms and no significant differences were found, although beta diversity results were significantly different in the treatment group between T0 and T1 time points. Streptococcus thermophilus, Bifidobacterium longum, Limosilactobacillus fermentum, and Ligilactobacillus salivarius species were identified as some of the most discriminant taxa positively associated with T1 samples. This preliminary study corroborates the relationship between intestinal microbiota and ASD recently described in the literature.

Treatments other than topical and systemic antibiotics are needed to restore the dysbiosis correlated with acne onset and evolution. In this view, probiotics and botanical extracts could represent a valid adjunctive therapeutic approach. The purpose of this study was to test the efficacy of a dietary supplement containing probiotics (Bifidobacterium breve BR03 DSM 16604, Lacticaseibacillus casei LC03 DSM 27537, and Ligilactobacillus salivarius LS03 DSM 22776) and botanical extract (lupeol from Solanum melongena L. and Echinacea extract) in subjects with mild to moderate acne over an 8-week study period.Monocentric, randomized, double-blind, four-arm, placebo-controlled clinical study involving 114 subjects.A significant (p 0.05) effect on the number of superficial inflammatory lesions was reported over the study period in the subjects taking the study agent (group II) (-56.67%), the botanical extracts (group III) (-40.00%), and the probiotics (group IV) (-38.89%) versus placebo (-10.00%). A significant (p 0.05) decrease in mean desquamation score, sebum secretion rate, and porphyrin mean count versus baseline was also reported, and the effect was most evident for group II. The analysis of log relative abundance after 4 and 8 weeks of treatment compared with baseline showed a significant (p 0.01) decrease in Cutibacterium acnes and S. aureus, along with a contextually and significant (p 0.05) increase in Staphylococcus epidermidis, especially in group II. No significant changes were reported for group I.The results from this study suggest that the administration of the dietary supplement under study was effective, safe, and well tolerated in subjects with mild to moderate acne and could represent a promising optional complement for the treatment of inflammatory acne as well as for control of acne-prone skin.

Goals The aim of this research was to evaluate whether micronized cells (MCs) from selected biotherapeutic bacteria have the ability to effectively modulate the polarization of monocyte/macrophage subpopulations to advantageously provide a first line of defense against infections. Background Inflammation is a reaction of the host to viral and bacterial infections with the physiological purpose of restoring tissue homeostasis. However, uncontrolled or unresolved inflammation can lead to tissue damage, giving rise to a plethora of chronic inflammatory diseases. The monocytes/macrophages play a key role in the initiation and resolution of inflammation through different activation programs. Study MCs were obtained from Bifidobacterium lactis BS01 strain using a Bioimmunizer extraction protocol. Monocytes were stimulated with the probiotic strain and/or MCs (10 mg/mL) for 24 hours and 5 days. Monocyte/macrophage differentiation was evaluated by cytometry analysis of surface markers and the activity of the 2 subpopulations on oxidative stress was assessed in an in vitro oxidative stress model with a spectrophotometric test. Results The MCs have been shown to modulate considerably the 2 subpopulations of human monocytes/macrophages, both the "patrolling subpopulation" and the "inflammatory subpopulation," thus highlighting a strong immunostimulatory effect. In addition, MCs are able to mitigate significantly the oxidative stress induced by homocysteine in an in vitro model. Conclusions Our findings suggest that MCs derived from the biotherapeutic strain BS01 could represent a possible therapy aimed to effectively prevent and/or cure viral, bacterial, fungal, or protozoal diseases, as well as prevent and/or treat inflammatory processes triggered by external pathogenic agents.

Goals The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. Background Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. Study Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39 /ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. Results The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. Conclusions On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.

Human beings harbor clusters of bacteria in different parts of the body, such as the surface or the deep layers of the skin, the mouth, the lungs, the intestine, the vagina, and all the surfaces exposed to the outer world. The majority of microbes resides in the gut, have a weighty influence on human physiology and nutrition and are vital for human life. There is growing evidence showing that the gut microbiota plays important roles in the maturation of the immune system and the protection against some infectious agents. In addition, there are several well-known effects of exercise on gut physiology. Exercise volume and intensity have been shown to exert an influence on gastrointestinal health status. An estimated 20% to 60% of athletes suffer from stress caused by excessive exercise and inadequate recovery. Supplementing the diet with prebiotics and/or probiotics able to improve the metabolic, immune, and barrier function can be a therapy for athletes. A recent study showed the effects of coadministration of 2 probiotic strains (Bifidobacterium breve BR03 and Streptococcus thermophilus FP4) on measures of skeletal muscle performance, damage, tension, and inflammation following a bout of strenuous exercise. Probiotic supplementation likely enhanced isometric average peak torque production from 24 to 72 hours into the recovery period following exercise. The active formulation also moderately increased resting arm angle at 24 and 48 hours following exercise. In conclusion, selected beneficial bacteria could positively affect athletes undergoing periods of intense training and may assist in the performance recovery.

Goals The aim of this research was to assess the antibacterial activity of Lactobacillus salivarius LS03 (DSM 22776) against Propionibacterium acnes and its anti-inflammatory properties by inhibiting P. acnes-induced interleukin-8 (IL-8) release. Background Acne is the most common skin disease, causing significant psychosocial problems for those afflicted. Currently available agents for acne treatment, such as oral antibiotics, have limited use. Thus, development of novel agents to treat this disease is needed. In the generation of inflammatory lesions, proliferation of P. acnes in the obstructed follicles is critical. The administration of beneficial microorganisms represents a promising approach for treating several skin alterations and can have many favorable effects. Study For the inhibition assay, P. acnes was spread on Propionibacter Isolation Agar Base plates, and LS03-soaked disks were placed directly on the agar surface. Peripheral blood mononuclear cells, isolated from healthy volunteers, were preincubated with phytohemagglutinin 1 μg/mL for 1 hour and stimulated with the probiotic strains for 24 hours to simulate an in vitro IL-8 release model. The IL-8 concentration in the supernatants was analyzed in duplicate using ELISA Kit. Results L. salivarius LS03 exerted a significant inhibitory capacity against the target pathogen strain. This antagonistic activity was primarily ascribable to the feature of LS03 strain of secreting active bacteriocins against P. acnes. Concerning the IL-8 analysis, 3 different L. salivarius strains were able to inhibit the release of this chemokine by 10% to 25%. Conclusions L. salivarius LS03 probiotic strain could be an alternative treatment to antibiotic/anti-inflammatory therapy in subjects presenting acne vulgaris.

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.

Background & aims: Variations in gut microbiota might impact metabolism leading to body weight excess. We assessed the impact of a probiotic supplementation in pediatric obesity on weight, metabolic alterations, selected gut microbial groups, and functionality. Methods: Cross-over, double-blind, randomized control trial (BIFI-OBESE trial; NCT03261466). 101 youths (6–18 years, Tanner stage ≥2) with obesity and insulin-resistance on diet were randomized to 2 × 109 CFU/AFU/day of Bifidobacterium breve BR03 (DSM 16604) and B. breve B632 (DSM 24706) (51) or placebo (50) for 8 weeks with a 4-weeks wash-out period. Results: All subjects (M/F 54/47) completed the first 8 weeks, and 82 (M/F 43/39) the last part without adverse events. Mixed-effects models revealed a carry-over effect on many variables in the entire study, narrowing the analysis to the first 8 weeks before the wash-out periods. All subjects improved metabolic parameters, and decreased weight and Escherichia coli counts. Probiotics improved insulin sensitivity at fasting (QUICKI, 0.013 CI95%0.0–0.03) and during OGTT (ISI, 0.654 CI95%-0.11–1.41). Cytokines, GLP1, and target microbial counts did not vary. Of 25 SCFAs, acetic acid and acetic acid pentyl-ester relative abundance remained stable in the probiotics, while increased in the placebo (p < 0.02). A signature of five butanoic esters identified three clusters, one of them had better glucose responses during probiotics. Conclusion: An 8 weeks treatment with B. breve BR03 and B632 had beneficial effects on insulin sensitivity in youths with obesity. Microbiota functionality could influence metabolic answers to probiotics. Long-term studies to confirm and enrich our findings are justified. Tailored probiotic treatments could be an additional strategy for obesity. Trial registration: NCT03261466.

A double-blind, placebo-controlled study was performed in a sample of geriatric patients treated with home enteral nutrition (HEN) to analyze the efficacy of a probiotic supplement Proxian&reg, which contains Lactiplantibacillus plantarum LP01 (LMG P-21021), Lentilactobacillus buchneri Lb26 (DSM 16341), Bifidobacterium animalis subsp. lactis BS01 (LMG P-21384), and is enriched with zinc (Zn) and selenium (Se), in reducing the incidence of infections and modulating inflammation. Thirty-two subjects were enrolled (mean age 79.7 &plusmn, 10.3 years), 16 in the intervention group, 16 controls. They received Proxian&reg, or placebo for 60 days. Patients were assessed at baseline (t0) and 60 (t1) and 90 (t2) days after the beginning. Infections were detected by information regarding their clinical manifestations and the incidence of antibiotic therapy. Levels of C-reactive protein (CRP) were measured to study inflammation. Information on bowel function, nutritional status and testimonials regarding the feasibility of administration of the product were collected. Differences between the two groups in number of infections (25% intervention group vs. 44% controls), antibiotic therapies (12% vs. 37%) and modulation of CRP levels (median CRP moved from 0.95 mg/L (t0), to 0.6 (t1) and 0.7 (t2) in intervention group vs. 0.7 mg/L, 0.5 and 0.7 in controls) did not reach statistical significance. No significant changes in bowel function and nutritional status were found. Caregivers&rsquo, adherence was 100%. Results of this &ldquo, IntegPRO&rdquo, study showed that Proxian&reg, is potentially safe, easy to administer and promising for further studies but it appears not to change the incidence of infections or modulate inflammation in elderly treated with HEN. The utility of Proxian&reg, in reducing the incidence of infections and modulating inflammation in these subjects needs to be investigated by a larger multi-center clinical trial, and by using additional analyses on inflammatory markers and markers of infections.

Celiac disease (CD) is a permanent intolerance to dietary protein, gluten, from wheat rye and barley. It occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and innate immune activation are responsible for breaking oral tolerance to gliadin, the gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several evidences show that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD, through the modulation of intestinal permeability and the regulation of the immune system. Here we show that gliadin induces a chronic ER stress condition in the small intestine of a CD mouse model and that the co-administration of probiotics efficiently attenuates both UPR and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, rebalancing the gut microbiota composition by probiotics administration might rep-resent a new strategy to treat CD affected patients.

Microbiota is a term coined to describe the population of bacteria, viruses and fungi that inhabit in symbiosis within a living host. A connection between unbalanced microbiota and chronic kidney disease has been established. In these patients, high levels of urea reach the intestine promoting the overgrowth of bacterial species that are prone to generate uremic toxins. Due to the high morbidity and mortality of this condition, a large number of therapeutic approaches to reduce inflammation and microbial uremic toxins have been proposed, with controversial results. A low protein diet, with a protein intake of 0.6–0.8 g/kg of body weight, is a useful and historically pursued option with this regard. The aim of our study is to evaluate, among patients with advanced renal failure not on dialysis, the synergic beneficial effects of this diet and the selected probiotics Bifidobacterium longum (mix DLBL) and Lactobacillus reuteri LRE02 (DSM 23878).

Recently, research has been deeply focusing on the role of the microbiota in numerous diseases, either affecting the skin or other organs. What it is well established is that its dysregulation promotes several cutaneous disorders (i.e. psoriasis and atopic dermatitis). To date, little is known about its composition, mediators and role in the genesis, progression and response to therapy of Non-Melanoma Skin Cancer (NMSC). Starting from a bibliographic study, we classified the selected articles into four sections: i) normal skin microbiota; ii)

Intestinal dysbiosis (changes in the gut commensal microbiome) is related to several ophthalmic diseases. The aim of this study was to verify whether oral specific probiotics can alter the clinical course of chalaziosis and its recurrence. A prospective comparative pilot study involving 26 children suffering from chalaziosis was conducted. Children were randomly divided into two groups. The first group received medical treatment (lid hygiene, warm compression and dexamethasone/tobramycin ointment for at least 20 days), and the second group received medical treatment plus a daily supplementation of oral probiotics (≥ 1 × 10^9 live cells of Streptococcus thermophilus ST10 (DSM 25246), ≥ 1 × 10^9 live cells of Lactococcus lactis LCC02 (DSM 29536) and ≥ 1 × 10^9 live cells of Lactobacillus delbrueckii subsp. bulgaricus (DSM 16606) with maltodextrin as the bulking agent (Probiotical S.p.A., Novara, Italy). All patients were evaluated at 2-week intervals for 3 months. If the lesion had not disappeared or decreased in size to 1 mm or less in diameter at the time of subsequent visits, the same procedure was repeated for another 3-month cycle. There was a significant difference in the time taken for complete resolution of the chalazion between the two groups in favour of the children receiving the probiotics. The treatment was not associated with any significant complications in either group. Trial registration: The trial was registered at clinical trials.gov under NCT04322500 on 25/03/2020 ("retrospectively registered").Conclusions: Modification of the intestinal microbiome with specific probiotics can alter the clinical course of chalaziosis in children by re-establishing intestinal and immune homeostasis. Probiotic supplementation can increase the effectiveness of traditional therapies by prompting the complete resolution of chalaziosis in a shorter amount of time, in an easy and feasible way. What is Known: • The intestinal microbiome plays a crucial role in several inflammatory diseases of the eye and is considered a therapeutic target. • Probiotics play a role in the prevention and treatment of different conditions in children. What is New: • In children probiotic supplementation is safe and effective. • Probiotic supplementation reduced the time required for complete resolution of the chalazion.

Monocytes are involved in innate immune surveillance, establishment and resolution on inflammation, and can polarize versus M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. The possibility to control and drive immune cells activity through plasma stimulation is therefore attractive. We focused on the effects induced by cold-atmospheric plasma on human primary monocytes and monocyte-derived macrophages. Monocytes resulted more susceptible than monocyte-derived macrophages to the plasma treatment as demonstrated by the increase in reactive oxygen (ROS) production and reduction of viability. Macrophages instead were not induced to produce ROS and presented a stable viability. Analysis of macrophage markers demonstrated a time-dependent decrease of the M1 population and a correspondent increase of M2 monocyte-derived macrophages (MDM). These findings suggest that plasma treatment may drive macrophage polarization towards an anti-inflammatory phenotype.

GOALS To determine the in vitro antimicrobial activity of selected Lactobacillus strains isolated from the feces of healthy humans against Klebsiella pneumoniae. BACKGROUND Klebsiella is ubiquitous in nature and may colonize the skin, the pharynx, or the gastrointestinal tract of humans. Despite the widespread use of antibiotic molecules with a broad spectrum in hospitalized patients, an increased overall load of klebsiellae as well as the subsequent development of multidrug-resistant strains able to synthesize extended-spectrum beta-lactamase have been registered. These strains are particularly virulent, express capsular-type K55, and have a considerable ability to propagate. STUDY The 4 strains Lactobacillus paracasei LPC01 (CNCM I-1390), Lactobacillus rhamnosus LR04 (DSM 16605), Bifidobacterium longum B2274 (DSM 24707), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were tested. The analysis was performed using both a disc-diffusion assay and the broth-dilution procedure, also including an evaluation of the supernatants obtained from a fresh broth culture of each bacterium. RESULTS L. delbrueckii subsp. delbrueckii LDD01 demonstrated the best inhibitory results among all the tested strains. The antibacterial activity of the supernatant was retained even after treatment with α-amylase and neutralization with NaOH 1N, thus suggesting the protein structure of the inhibitory molecule. In contrast, it was completely lost after treatment with proteinase K. CONCLUSIONS Overall results suggest that the inhibitory effect of L. delbrueckii subsp. delbrueckii LDD01 should be attributed to the production of a bacteriocin. This strain may be prospectively useful for strengthening probiotic formulations and possibly counteract infections by K. pneumoniae in humans.

Tocilizumab, etanercept and abatacept are biological drugs used in the therapy of Rheumatoid Arthritis (RA). Their mechanism of action is well documented but their direct effects on human monocytes/macrophages have not been fully investigated. The objective of this study was to evaluate in vitro the influence of these drugs on monocytes/macrophages from healthy volunteers. Human monocytes were isolated from healthy anonymous volunteers and cultured as such or differentiated to monocyte-derived macrophages (MDMs). The effect of tocilizumab, etanercept and abatacept (at concentrations similar to those in plasma of patients) on superoxide anion production, matrix metalloproteinase-9 (MMP-9) gene expression and activity, Peroxisome Proliferator-Activated Receptor (PPAR)γ expression and cell phenotype was evaluated. Exposure of monocytes/macrophages to tocilizumab, etanercept or abatacept resulted in a significant decrease of the PMA-induced superoxide anion production. Interestingly, the expression of PPARγ was significantly increased only by tocilizumab, while etanercept was the only one able to significantly reduce MMP-9 gene expression and inhibit the LPS-induced MMP-9 activity in monocytes. When etanercept and abatacept were added to the differentiating medium, both significantly reduced the amount of CD206(+)MDM. This study demonstrates that etanercept, abatacept and tocilizumab affect differently human monocytes/macrophages. In particular, the IL-6 antagonist tocilizumab seems to be more effective in inducing an anti-inflammatory phenotype of monocytes/macrophages compared to etanercept and abatacept, also in light of the up-regulation of PPARγ whose anti-inflammatory effects are well recognised.

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

The establishment of the beneficial interactions between the host and its microbiota is essential for the correct functioning of the organism, since microflora alterations can lead to many diseases. Probiotics improve balanced microbial communities, exerting substantial health-promoting effects. Here we monitored the molecular outcomes, obtained by gut microflora modulation through probiotic treatment, on human urine and serum metabolic profiles, with a metabolomic approach. Twenty-two subjects were enrolled in the study and administered with two different probiotic types, both singularly and in combination, for 8 weeks. Urine and serum samples were collected before and during the supplementation and were analyzed by nuclear magnetic resonance (NMR) spectroscopy and statistical analyses. After eight weeks of treatment, probiotics deeply influence the urinary metabolic profiles of the volunteers, without significantly altering their single phenotypes. Anyway, bacteria supplementation tends to reduce the differences in metabolic phenotypes among individuals. Overall, the effects are recipient-dependent, and in some individuals, robust effects are already well visible after four weeks. Modifications in metabolite levels, attributable to each type of probiotic administration, were also monitored. Metabolomic analysis of biofluids turns out to be a powerful technique to monitor the dynamic interactions between the microflora and the host, and the individual response to probiotic assumption.

The number of bacterial cells is currently recognized as the most important parameter for the efficacy and quality of finished probiotic or live biotherapeutic products (LBP). Cell enumeration is generally performed by culture-dependent methodologies like plate count (PC). These techniques are able to reveal the number of viable cells able to replicate and generate a colony. However, they are limited by their dependence on the combination of culture conditions (e.g. nutrients, temperature) selected for cell recovery. Additionally, they do not provide information on the heterogeneity of a bacterial culture, namely they do not detect the cells in a viable but not cultivable (VBNC) status. Flow-cytometry (FC) is a culture-independent methodology having the potential to enumerate selectively live and damaged or dead cells. FC relies on the use of specific probes for different cell targets (e.g. membrane, enzymes) to unveil information on the cell structure and physiological statuses within a bacterial population. In this context, we monitored three batches of freeze-dried Lactobacillus rhamnosus GG (ATCC 53103) during a 3 year of storage at different conditions of temperature and relative humidity, according to ICH guidelines, by means of PC and FC. The Arrhenius model was applied to assess the suitability of the model to predict the mortality of probiotic cells in finished products. The higher destruction rate (k) obtained by PC data compared to FC data suggests a faster reduction of cultivability compared to membrane integrity, probably representing a dynamic shift of the bacterial population into a VBNC/dormant status during storage time. Interestingly, this mechanistic approach works both for PC and FC methodologies increasing the chances to monitor biological phenomenon within a mathematical modelling. The combined use of PC and FC shed lights on the true bacterial potency within a closed system like a finished product and the complexity of its heterogeneity.

Goals This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and Gardnerella vaginalis to weigh its potential effectiveness even in mixed vaginal infections. Background A wide female population is suffering from various vulvovaginal infections. These diseases are often associated with a decrease in the concentration of Lactobacilli in the vagina. Mixed vaginal infections represent >20% of women with vulvovaginal infection. Study LF5 strain was cocultured in De Man, Rogosa and Sharpe with Candida according to a 1:100 ratio in favor of the yeast. Each culture was sampled after 24 hours of incubation for the selective enumeration of the yeasts performed on yeast extract glucose chloramphenicol agar medium.The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants of L. fermentum LF5 ranging from 5% to 20% was quantified by means of optical density at 600 nm (OD600). Results L. fermentum LF5 demonstrated the ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms.Furthermore, L. fermentum LF5 showed a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth. Conclusions The biotherapeutic LF5 could be the only documented strain effective in mixed forms. For this purpose, a human clinical trial is in progress.

Background Recent preclinical studies suggest that dysfunction of gastrointestinal tract may play a role in amyotrophic lateral sclerosis (ALS) pathogenesis through a modification of the gut microbiota brain axis. Our study is the first focused on microbiota analysis in ALS patients. Aim Our aim was to study the main human gut microbial groups and the overall microbial diversity in ALS and healthy subjects. Moreover we have examined the influence of a treatment with a specific bacteriotherapy composed of Lactobacillus strains (Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus salivarius) acting on the gastrointestinal barrier. Methods We enrolled 50 ALS patients and 50 healthy controls, matched for sex, age, and origin. Fecal samples were used for total genomic DNA extraction. Enterobacteria, Bifidobacterium spp., Lactobacillus spp., Clostridium sensu stricto, Escherichia coli and yeast were quantified using quantitative polymerase chain reaction approach. Denaturing gradient gel electrophoresis analyses were performed to investigate total eubacteria and yeasts populations. Patients were randomized to double-blind treatment either with microorganisms or placebo for 6 months and monitored for clinical progression and microbiota composition. Results The comparison between ALS subjects and healthy group revealed a variation in the intestinal microbial composition with a higher abundance of E. coli and enterobacteria and a low abundance of total yeast in patients. Polymerase chain reaction denaturing gradient gel electrophoresis analysis showed a cluster distinction between the bacterial profiles of ALS patients and the healthy subjects. The complexity of the profiles in both cases may indicate that a real dysbiosis status is not evident in the ALS patients although differences between healthy and patients exist. The effects of the progression of the disease and of the bacteriotherapy on the bacterial and yeast populations are currently in progress. Conclusions Our preliminary results confirm that there is a difference in the microbiota profile in ALS patients.

Neurokinin 1 receptors (NK-1R) have been involved in several psychiatric disorders including major depression, but less is known for bipolar disorder (BD).We compared NK-1R expression and Substance P (SP) ability to induce NF-κB activation in monocytes from BD patients and healthy donors (HD), also looking for the effects of tobacco smoke. After informed written consent, 20 euthymic BD patients, either bipolar type 1 (BDI) or type 2 (BDII), and 14 age-matched healthy donors (HD) were enrolled. NK-1R expression in monocytes was evaluated by Western blot and expressed as the ratio between NK-1R and Na(+)/K(+)-ATPase protein expressions. NF-κB activation was assessed by measuring the nuclear content of the p50 subunit (ELISA kit).NK-1R expression was significantly reduced (P0.001) in monocytes from BD patients as compared to HD, with no major differences between BDI and BDII patients. Tobacco smoke enhanced NK-1R expression in HD, but not in BD patients. Un-stimulated monocytes from BD patients presented a constitutively higher (P0.05) content of nuclear p50 subunit as compared to HD. SP and an NK-1R agonist induced NF-κB activation, with a higher effect in HD: this effect was receptor-mediated as it was abrogated by an NK-1R antagonist.As a pilot study enrolling 20 BD patients, an obvious limitation is the sample size.Our results show the existence of a relevant alteration in NK-1R expression in BD patients and further suggest SP involvement in BD, so improving our understanding of the underlying mechanisms of this disease.