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Heart failure (HF) is still characterized by high mortality rates, despite the progress achieved in terms of treatment options. With regard to the treatment of HF with reduced ejection fraction (HFrEF), the 2016 European Society of Cardiology guidelines included in the therapeutic algorithm the angiotensin receptor-neprilysin inhibitor class, whose efficacy in modifying patient prognosis has been extensively proven in many clinical studies. Sacubitril/valsartan, the only representative of this drug class, can effectively affect the natural history of HF, thus reducing cardiovascular mortality (sudden death and death due to worsening cardiac function), total mortality, as well as first and recurrent hospitalization events, by improving renal function, cardiac remodeling, functional capacity and the patient's health-related quality of life.The purpose of this article is to analyze the different phases of the journey of patients with HFrEF (first general practitioner consultation; admission to the emergency department and subsequent hospitalization; referral to a specialist HF clinic) and promotion of a networking approach involving the general practitioner, the hospital and the HF specialist based on common pre-defined diagnostic and therapeutic protocols, that meets patient needs at all stages of the disease (case-specific dosing assessment, drug titration before follow-up and prevention of adverse events).

People with cryoglobulinaemic vasculitis (CV) have an increased risk of infections, attributed to different causes: impairment of the immune system due to the disease itself, comorbidities, and immunosuppressive therapy. Therefore, these patients may be at high risk for a more severe course of COVID-19, including hospitalisation and death. Concerns about efficacy, immunogenicity and safety of vaccines, as well as doubts, not yet fully clarified in patients with systemic autoimmune diseases, represent other important factors for a low vaccination rate in people with (CV). Indeed, providing an expert position on the issues related to SARS-CoV-2 vaccination in patients suffering from CV is of critical relevance in order to help both patients and clinicians who are treating them in making the best choice in each case. A multidisciplinary task force of the Italian Group for the Study of Cryoglobulinaemia (GISC) was convened, and through a Delphi technique produced provisional recommendations regarding SARS-CoV-2 vaccination in cryoglobulinaemic patients.

Based on serological and molecular evidence of hepatitis C virus (HCV) infection in a significant proportion of patients with mixed cryoglobulinemia (MC), a direct association between HCV and MC has been suggested. The goal of the present study was to investigate the role played by HCV and by the immune response to the virus in the pathogenesis of mixed cryoglobulinemia.A competitive reverse transcription polymerase chain reaction was employed to evaluate the concentrations of specific HCV RNA sequences in different clinical specimens (plasma, sera, cryoprecipitates, bone marrow and peripheral blood cells). Using recombinant and synthetic peptides covering the HCV core, envelope 1 (E1) and nonstructural regions 4 (NS4) and 5 (NS5), the humoral immune response in a group of MC patients was assessed with an enzyme-linked immunosorbent assay. Natural killer (NK) cell activity was estimated using a 4 hr 51 Cr release assay.Quantitation of the RNA molecules in the biological samples confirmed an increased virion concentration in cryoprecipitates from 13/15 patients with mixed cryoglobulinemia. Analysis of the humoral immune response against the synthetic peptides suggested a distinct response to HCV antigens in MC patients when compared to patients with HCV infection but without serological evidence of cryoglobulinemia. Unstimulated NK cell functioning was below the normal range in all patients tested. However, peripheral blood mononuclear cells showed no enhancement of NK activity by the interferon inducer polyinosinic acid:polycytidilic acid. Enhancement by interferon-alpha was normal, suggesting an impairment in interferon production.The quantitative data are in line with the hypothesis of a direct or indirect role of HCV in mixed cryoglobulinemia. The abnormal immune response could be involved in the onset and persistence of HCV infection, and possibly in the appearance of cryoglobulinemia.

We have investigated the distribution of TGF beta using antibodies specific for its intracellular and extracellular forms in full-thickness biopsies of patients with SSc, primary Raynaud's phenomenon (PRP), systemic lupus erythematosus (SLE), and from normal subjects. Nine of 11 SSc biopsies demonstrated intracellular TGF beta in endothelial cells while only 6 exhibited extracellular TGF beta. Endothelial cells in skin biopsies of all PRP patients displayed both intracellular and extracellular TGF beta. All other control biopsies were negative. In patients with PRP, some positively staining fibroblasts were found scattered throughout the dermis. Lastly, extracellular TGF beta was localized in the papillary dermis of PRP and SSc biopsies and in all the dermal layers of SLE patients. No significant staining of TGF beta was observed in the endothelial cells, fibroblasts, or in the extracellular matrix of the majority of biopsies from normal subjects. These data suggest that TGF beta may be one of the cytokines involved in the early stages of pathogenesis of SSc, and that endothelial cells in SSc and PRP may be a source and/or a target of TGF beta.

The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface. © 2012 .

Commonly used non-antibiotic drugs have been associated with changes in gut microbiome composition, paving the way for the possibility of repurposing FDA-approved molecules as next-generation microbiome therapeutics. Herein, we developed and validated an ex vivo high-throughput screening platform─the mini gut model─to underpin human gut microbiome response to molecular modulators. Ten FDA-approved compounds, selected based on maximum structural diversity of molecular fingerprints, were screened against the gut microbiome of five healthy subjects to characterize the ability of human-targeted drugs to modulate the human gut microbiome network. Three compounds, THIP hydrochloride, methenamine, and mesna, have shown promise as novel gut microbiome therapeutics in light of their capability of promoting health-associated features of the gut microbiome. Our findings provide a resource for future research on drug-microbiome interactions and lay the foundation for a new era of more precise gut microbiome modulation through drug repurposing, aimed at targeting specific dysbiotic events.

Antibiotic resistance (AR) is indisputably a major health threat which has drawn much attention in recent years. In particular, the gut microbiome has been shown to act as a pool of AR genes, potentially available to be transferred to opportunistic pathogens. Herein, we investigated for the first time changes in the human gut resistome during aging, up to extreme longevity, by analyzing shotgun metagenomics data of fecal samples from a geographically defined cohort of 62 urban individuals, stratified into four age groups: young adults, elderly, centenarians, and semisupercentenarians, i.e., individuals aged up to 109 years. According to our findings, some AR genes are similarly represented in all subjects regardless of age, potentially forming part of the core resistome. Interestingly, aging was found to be associated with a higher burden of some AR genes, including especially proteobacterial genes encoding multidrug efflux pumps. Our results warn of possible health implications and pave the way for further investigations aimed at containing AR accumulation, with the ultimate goal of promoting healthy aging. IMPORTANCE Antibiotic resistance is widespread among different ecosystems, and in humans it plays a key role in shaping the composition of the gut microbiota, enhancing the ecological fitness of certain bacterial populations when exposed to antibiotics. A considerable component of the definition of healthy aging and longevity is associated with the structure of the gut microbiota, and, in this regard, the presence of antibiotic-resistant bacteria is critical to many pathologies that come about with aging. However, the structure of the resistome has not yet been sufficiently elucidated. Here, we show distinct antibiotic resistance assets and specific microbial consortia characterizing the human gut resistome through aging.

Multiple sclerosis (MS) is a neurodegenerative inflammatory condition mediated by autoreactive immune processes. Due to its potential to influence host immunity and gut-brain communication, the gut microbiota has been suggested to be involved in the onset and progression of MS. To date, there is no definitive cure for MS, and rehabilitation programs are of the utmost importance, especially in the later stages. However, only a few people generally participate due to poor support, knowledge, and motivation, and no information is available on gut microbiota changes. Herein we evaluated the potential of a brief high-impact multidimensional rehabilitation program (B-HIPE) in a leisure environment to affect the gut microbiota, mitigate MS symptoms and improve quality of life. B-HIPE resulted in modulation of the MS-typical dysbiosis, with reduced levels of pathobionts and the replenishment of beneficial short-chain fatty acid producers. This partial recovery of a eubiotic profile could help counteract the inflammatory tone typically observed in MS, as supported by reduced circulating lipopolysaccharide levels and decreased populations of pro-inflammatory lymphocytes. Improved physical performance and fatigue relief were also found. Our findings pave the way for integrating clinical practice with holistic approaches to mitigate MS symptoms and improve patients’ quality of life.

Despite well-established knowledge of the role of diet and the geographic effect on the gut microbiota of human populations, the temporal dynamics of the individual microbiota profile across changes associated with intercontinental short residence are still far from being understood. This pilot study sought to provide insights into the trajectory of the gut microbiota of an individual during a two-month stay in Italy and a subsequent two-month stay in Nigeria, by 16S rRNA gene sequencing and inferred metagenomics. The gut microbiota underwent massive but temporary changes, both taxonomically and based on predicted functionality. The faecal microbiota associated with the short stay in Italy progressively lost diversity and showed a dominance of Firmicutes, while after returning to Nigeria, the microbial community quickly regained the typical profile, in terms of biodiversity and bacterial signatures of traditional lifestyle, i.e., Prevotella and Treponema. Predicted pathways involved in glycolysis, fermentation and N-acetylneuraminate degradation were enriched during the subsequent two-month stay in Nigeria, whereas pathways associated with amino acid and peptidoglycan synthesis and maturation became over-represented during short stay in Italy. Our findings stress the plasticity of the individual gut microbiota even during a short-term travel, with loss/gain of taxonomic and functional features that mirror those of the gut microbiota of indigenous people dwelling therein.

Aims: To assess the effects of walnuts on cardiometabolic outcomes in obese people and to explore the underlying mechanisms using novel methods including metabolomic, lipidomic, glycomic and microbiome analysis, integrated with lipid particle fractionation, appetite-regulating hormones and haemodynamic measurements. Materials and Methods: A total of 10 obese individuals were enrolled in this cross-over, randomized, double-blind, placebo-controlled clinical trial. The participants had two 5-day inpatient stays, during which they consumed a smoothie containing 48 g walnuts or a macronutrient-matched placebo smoothie without nuts, with a 1-month washout period between the two visits. Results: Walnut consumption improved aspects of the lipid profile; it reduced fasting small and dense LDL particles (P < 0.02) and increased postprandial large HDL particles (P < 0.01). Lipoprotein insulin resistance score, glucose and the insulin area under the curve (AUC) decreased significantly after walnut consumption (P < 0.01, P < 0.02 and P < 0.04, respectively). Consuming walnuts significantly increased 10 N-glycans, with eight of them carrying a fucose core. Lipidomic analysis showed a robust reduction in harmful ceramides, hexosylceramides and sphingomyelins, which have been shown to mediate effects on cardiometabolic risk. The peptide YY AUC significantly increased after walnut consumption (P < 0.03). No major significant changes in haemodynamic or metabolomic analysis or in microbiome host health-promoting bacteria such as Faecalibacterium were found. Conclusions: These data provide a more comprehensive mechanistic perspective of the effect of dietary walnut consumption on cardiometabolic variables. Lipidomic and lipid nuclear magnetic resonance spectroscopy analysis showed an early but significant reduction in ceramides and other atherogenic lipids with walnut consumption, which may explain the longer-term benefits of walnuts or other nuts on insulin resistance, cardiovascular risk and mortality.

Background Modern metagenomic analysis of complex microbial communities produces large amounts of sequence data containing information on the microbiome in terms of bacterial, archaeal, viral and eukaryotic composition. The bioinformatics tools available are mainly devoted to profiling the bacterial and viral fractions and only a few software packages consider fungi. As the human fungal microbiome (human mycobiome) can play an important role in the onset and progression of diseases, a comprehensive description of host-microbiota interactions cannot ignore this component. Results HumanMycobiomeScan is a bioinformatics tool for the taxonomic profiling of the mycobiome directly from raw data of next-generation sequencing. The tool uses hierarchical databases of fungi in order to unambiguously assign reads to fungal species more accurately and > 10,000 times faster than other comparable approaches. HumanMycobiomeScan was validated using in silico generated synthetic communities and then applied to metagenomic data, to characterize the intestinal fungal components in subjects adhering to different subsistence strategies. Conclusions Although blind to unknown species, HumanMycobiomeScan allows the characterization of the fungal fraction of complex microbial ecosystems with good performance in terms of sample denoising from reads belonging to other microorganisms. HumanMycobiomeScan is most appropriate for well-studied microbiomes, for which most of the fungal species have been fully sequenced. This released version is functionally implemented to work with human-associated microbiota samples. In combination with other microbial profiling tools, HumanMycobiomeScan is a frugal and efficient tool for comprehensive characterization of microbial ecosystems through shotgun metagenomics sequencing. Electronic supplementary material The online version of this article (10.1186/s12864-019-5883-y) contains supplementary material, which is available to authorized users.

This study evaluates the transcriptional profiles of genes related to physiological responses in digestive glands (DG) of Mytilus galloprovincialis under the influence of seasonal changes of environmental variables, gender bias, and gonadal development. Composition of the DG microbiome was also explored. Mussels were collected across 7 months encompassing 3 seasons from a farm in the Northwestern Adriatic Sea. All gene products showed complex transcriptional patterns across seasons. Salinity, surface oxygen and transparency significantly correlate with transcriptional profiles of males, whereas in females temperature and gonadal maturation mostly explained the observed transcriptional changes. Seasonal variations and gender-specific differences were observed in DG microbiome composition, with variations resembling metabolic accommodations likely facing season progression and reproductive cycle. Results provide baseline information to improve actual monitoring strategies of mussel farming conditions and forecast potential detrimental impacts of climatological/environmental changes in the study area.

After spinal cord injury (SCI), patients face many physical and psychological issues including intestinal dysfunction and comorbidities, strongly affecting quality of life. The gut microbiota has recently been suggested to influence the course of the disease in these patients. However, to date only two studies have profiled the gut microbiota in SCI patients, months after a traumatic injury. Here we characterized the gut microbiota in a large Italian SCI population, within a short time from a not only traumatic injury. Feces were collected within the first week at the rehabilitation center (no later than 60 days after SCI), and profiled by 16S rRNA gene-based next-generation sequencing. Microbial profiles were compared to those publicly available of healthy age- and gender-matched Italians, and correlated to patient metadata, including type of SCI, spinal unit location, nutrition and concomitant antibiotic therapies. The gut microbiota of SCI patients shows distinct dysbiotic signatures, i.e. increase in potentially pathogenic, pro-inflammatory and mucus-degrading bacteria, and depletion of short-chain fatty acid producers. While robust to most host variables, such dysbiosis varies by lesion level and completeness, with the most neurologically impaired patients showing an even more unbalanced microbial profile. The SCI-related gut microbiome dysbiosis is very likely secondary to injury and closely related to the degree of completeness and severity of the lesion, regardless of etiology and time interval. This microbial layout could variously contribute to increased gut permeability and inflammation, potentially predisposing patients to the onset of severe comorbidities.

Enteral Nutrition (EN) is recommended as first line nutritional support for patients undergoing Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT), but only few studies exist in the literature which compare EN to Parenteral Nutrition (PN) in the paediatric population.Forty-two consecutive paediatric patients undergoing allo-HSCT at our referral centre between January 2016 and July 2019 were evaluated. Post-transplant and nutritional outcomes of patients receiving EN for more than 7 days (EN group, n = 14) were compared with those of patients receiving EN for fewer than 7 days or receiving only PN (PN group, n = 28). In the EN group, a reduced incidence of Blood Stream Infections (BSI) was observed (p = 0.02) (n = 2 vs. n = 15; 14.3% vs. 53.6%). The type of nutritional support was also the only variable independently associated with BSI in the multivariate analysis (p = 0.03). Platelet engraftment was shorter in the PN group than in the EN group for a threshold of > 20*109/L (p = 0.04) (23.1 vs 35.7 days), but this correlation was not confirmed with a threshold of > 50*109/L. The Body Mass Index (BMI) and the BMI Z-score were no different in the two groups from admission to discharge.Our results highlight that EN is a feasible and nutritionally adequate method of nutritional support for children undergoing allo-HSCT in line with the present literature. Future functional studies are needed to better address the hypothesis that greater intestinal eubyosis maintained with EN may explain the observed reduction in BSI.

Hematopoietic stem cell transplantation (HSCT) is the first-line immunotherapy to treat several hematologic disorders, although it can be associated with many complications reducing the survival rate, such as acute graft-versus-host disease (aGvHD) and infections. Given the fundamental role of the gut microbiome (GM) for host health, it is not surprising that a suboptimal path of GM recovery following HSCT may compromise immune homeostasis and/or increase the risk of opportunistic infections, with an ultimate impact in terms of aGvHD onset. Traditionally, the first nutritional approach in post-HSCT patients is parenteral nutrition (PN), which is associated with several clinical adverse effects, supporting enteral nutrition (EN) as a preferential alternative. The aim of the study was to evaluate the impact of EN vs. PN on the trajectory of compositional and functional GM recovery in pediatric patients undergoing HSCT. The GM structure and short-chain fatty acid (SCFA) production profiles were analyzed longitudinally in twenty pediatric patients receiving HSCT&mdash, of which, ten were fed post-transplant with EN and ten with total PN. According to our findings, we observed the prompt recovery of a structural and functional eubiotic GM layout post-HSCT only in EN subjects, thus possibly reducing the risk of systemic infections and GvHD onset.

Accumulating literature is providing evidence that the gut microbiota is involved in metabolic disorders, but the question of how to effectively modulate it to restore homeostasis, especially in the elderly, is still under debate. In this study, we profiled the intestinal microbiota of 20 elderly obese women (EO) at the baseline (T0), after 15 days of hypocaloric Mediterranean diet administered as part of a nutritional-metabolic rehabilitation program for obesity (T1), and after a further 15 days of the same diet supplemented with a probiotic mix (T2). Fecal samples were characterized by Illumina MiSeq sequencing of the 16S rRNA gene. The EO microbiota showed the typical alterations found in obesity, namely, an increase in potential pro-inflammatory components (i.e., Collinsella) and a decrease in health-promoting, short-chain fatty acid producers (i.e., Lachnospiraceae and Ruminococcaceae members), with a tendency to reduced biodiversity. After 15 days of the rehabilitation program, weight decreased by (2.7 &plusmn, 1.5)% and the gut microbiota dysbiosis was partially reversed, with a decline of Collinsella and an increase in leanness-related taxa. During the next 15 days of diet and probiotics, weight dropped further by (1.2 &plusmn, 1.1)%, markers of oxidative stress improved, and Akkermansia, a mucin degrader with beneficial effects on host metabolism, increased significantly. These findings support the relevant role of a correct dietetic approach, even in the short term, to modulate the EO gut microbiota towards a metabolic health-related configuration, counteracting the increased risk of morbidity in these patients.

Background Advances in bioinformatics recently allowed for the recovery of ‘metagenomes assembled genomes’ from human microbiome studies carried on with shotgun sequencing techniques. Such approach is used as a mean to discover new unclassified metagenomic species, putative biological entities having distinct metabolic traits. Results In the present analysis we compare 400 genomes from isolates available on NCBI database and 10,000 human gut metagenomic species, screening all of them for the presence of a minimal set of core functionalities necessary, but not sufficient, for life. As a result, the metagenome-assembled genomes resulted systematically depleted in genes encoding for essential functions apparently needed to support autonomous bacterial life. Conclusions The relevant degree of lacking core functionalities that we observed in metagenome-assembled genomes raises some concerns about the effective completeness of metagenome-assembled genomes, suggesting caution in extrapolating biological information about their metabolic propensity and ecology in a complex environment like the human gastrointestinal tract.

The gut microbiome of pediatric patients undergoing allo-hematopoietic stem cell transplantation (HSCT) has recently been considered as a potential reservoir of antimicrobial resistance, with important implications in terms of patient mortality rate. By means of shotgun metagenomics, here we explored the dynamics of the gut resistome – i.e. the pattern of antibiotic resistance genes provided by the gut microbiome – in eight pediatric patients undergoing HSCT, half of whom developed acute Graft-versus-Host Disease (aGvHD). According to our findings, the patients developing aGvHD are characterized by post-HSCT expansion of their gut resistome, involving the acquisition of new resistances, as well as the consolidation of those already present before HSCT. Interestingly, the aGvHD-associated bloom in resistome diversity is not limited to genes coding for resistance to the antibiotics administered along the therapeutic course, but rather involves a broad pattern of different resistance classes, including multidrug resistance, as well as resistance to macrolides, aminoglycosides, tetracyclines and beta-lactams. Our data stress the relevance of mapping the gut resistome in HSCT pediatric patients to define the most appropriate anti-infective treatment post HSCT.

The modern Paleolithic diet (MPD), featured by the consumption of vegetables, fruit, nuts, seeds, eggs, fish and lean meat, while excluding grains, dairy products, salt and refined sugar, has gained substantial public attention in recent years because of its potential multiple health benefits. However, to date little is known about the actual impact of this dietary pattern on the gut microbiome (GM) and its implications for human health. In the current scenario where Western diets, low in fiber while rich in industrialized and processed foods, are considered one of the leading causes of maladaptive GM changes along human evolution, likely contributing to the increasing incidence of chronic non-communicable diseases, we hypothesize that the MPD could modulate the Western GM towards a more “ancestral” configuration. In an attempt to shed light on this, here we profiled the GM structure of urban Italian subjects adhering to the MPD, and compared data with other urban Italians following a Mediterranean Diet (MD), as well as worldwide traditional hunter-gatherer populations from previous publications. Notwithstanding a strong geography effect on the GM structure, our results show an unexpectedly high degree of biodiversity in MPD subjects, which well approximates that of traditional populations. The GM of MPD individuals also shows some peculiarities, including a high relative abundance of bile-tolerant and fat-loving microorganisms. The consumption of plant-based foods–albeit with the exclusion of grains and pulses–along with the minimization of the intake of processed foods, both hallmarks of the MPD, could therefore contribute to partially rewild the GM but caution should be taken in adhering to this dietary pattern in the long term.

Gut microbiota, the specific microbial community of the gastrointestinal tract, by means of the production of microbial metabolites provides the host with several functions affecting metabolic and immunological homeostasis. Insights into the intricate relationships between gut microbiota and the host require not only the understanding of its structure and function but also the measurement of effector molecules acting along the gut microbiota axis. This article reviews the literature on targeted chromatographic approaches in analysis of gut microbiota specific metabolites in feces as the most accessible biological matrix which can directly probe the connection between intestinal bacteria and the (patho)physiology of the holobiont. Together with a discussion on sample collection and preparation, the chromatographic methods targeted to determination of some classes of microbiota-derived metabolites (e.g., short-chain fatty acids, bile acids, low molecular masses amines and polyamines, vitamins, neurotransmitters and related compounds) are discussed and their main characteristics, summarized in Tables.

Background (Trans)-3,7-Dimethyl-2,6-octadien-1-ol, commonly called geraniol (Ge-OH), is an acyclic monoterpene alcohol with well-known anti-inflammatory and antimicrobial properties. Ge-OH is a non-toxic compound classified as Generally Recognized As Safe (GRAS) by the US Food and Drug Administration and the European Food Security Agency. Methods Ge-OH was orally administered at a maximum daily dose of 8 mg kg(− 1) body weight for four weeks in a delayed release formulation capable of reaching the colon. Fecal microbiota and blood cytokines were analyzed before and after Ge-OH treatment, as well as IBS symptomatology by using Visual Analogue Scale (VAS-IBS). Results The results show that orally administered Ge-OH is a powerful modulator of the intestinal microbial ecosystem, capable of leading to increased relative abundances of Collinsella and especially Faecalibacterium, a well-known health-promoting butyrate producer consistently found to be decreased in IBS patients. Moreover, Ge-OH strongly improved the clinical symptoms of colitis by significantly reducing the score recorded by the VAS-IBS questionnaire. Clinical improvement was associated with a significant reduction in the circulating MIP-1β, a chemokine found to be increased in several IBS patients. Conclusion Ge-OH could be a powerful component for food supplement targeted to the treatment of IBS patients. Trial registration ISRCTN47041881, retrospectively registered on 19th July 2018.

Therapeutic hypothermia is today used in several clinical settings, among them the gut related diseases that are influenced by ischemia/reperfusion injury. This perspective paved the way to the study of hibernation physiology, in natural hibernators, highlighting an unexpected importance of the gut microbial ecosystem in hibernation and torpor. In natural hibernators, intestinal microbes adaptively reorganize their structural configuration during torpor, and maintain a mutualistic configuration regardless of long periods of fasting and cold temperatures. This allows the gut microbiome to provide the host with metabolites, which are essential to keep the host immunological and metabolic homeostasis during hibernation. The emerging role of the gut microbiota in the hibernation process suggests the importance of maintaining a mutualistic gut microbiota configuration in the application of therapeutic hypothermia as well as in the development of new strategy such as the use of synthetic torpor in humans. The possible utilization of tailored probiotics to mold the gut ecosystem during therapeutic hypothermia can also be taken into consideration as new therapeutic strategy.

The High Taxonomic Fingerprint (HTF)-Microbi.Array is a fully validated phylogenetic microarray platform for a high taxonomic level characterization of the human gut microbiota. However, suffering from PCR-dependent biases in Bifidobacterium quantification, this tool is less appropriate when utilized for the characterization of the Bifidobacterium -dominated gut microbiota of breast-fed infants. To overcome this, we implemented a new combined approach based on HTF-Microbi.Array and qPCR for a reliable fingerprint of the infant-type microbiota. This methodology was applied in a preliminary comparative study of the faecal microbiota of eight breast-fed infants, aged 2–6 months, and five young adults. Whereas the adult gut microbiota was largely dominated by Firmicutes and Bacteroidetes , the infant-type community was mainly dominated by Bifidobacterium , with Enterobacteriaceae as the second dominant component. In accordance with the most recent literature in the field, the obtained microbiota fingerprints properly depicted the adult- and the infant-type microbiota, demonstrating the reliability of the HTF-Microbi.Array/qPCR combined approach in reflecting the peculiarities of the two intestinal microbial ecosystems.

The ageing process affects the human gut microbiota phylogenetic composition and its interaction with the immune system. Age-related gut microbiota modifications are associated with immunosenescence and inflamm-ageing in a sort of self-sustaining loop, which allows the placement of gut microbiota unbalances among both the causes and the effects of the inflamm-ageing process. Even if, up to now, the link between gut microbiota and the ageing process is only partially understood, the gut ecosystem shows the potential to become a promising target for strategies able to contribute to the health status of older people. In this context, the consumption of pro/prebiotics may be useful in both prevention and treatment of age-related pathophysiological conditions, such as recovery and promotion of immune functions, i.e. adjuvant effect for influenza vaccine, and prevention and/or alleviation of common “winter diseases”, as well as constipation and Clostridium difficile -associated diarrhoea. Moreover, being involved in different mechanisms which concur in counteracting inflammation, such as down-regulation of inflammation-associated genes and improvement of colonic mucosa conditions, probiotics have the potentiality to be involved in the promotion of longevity.

Advances in biological techniques have potentiated great progresses in understanding the interaction between human beings and the ∼10 to 100 trillion microbes living in their gastrointestinal tract: gut microbiota (GM). In this review, we describe recent emerging data on the role of GM in hematopoietic stem cell transplantation, with a focus on immunomodulatory properties in the immune system recovery and the impact in the development of the main complications, as GvHD and infections.

Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis -plasmin(ogen) interaction in the human gastrointestinal tract.

Objective The objective of this review was to define a core set of recommendations for the treatment of HCV-associated mixed cryoglobulinemia syndrome (MCS) by combining current evidence from clinical trials and expert opinion. Methods Expert physicians involved in studying and treating patients with MCS formulated statements after discussing the published data. Their attitudes to treatment approaches (particularly those insufficiently supported by published data) were collected before the consensus conference by means of a questionnaire, and were considered when formulating the statements. Results An attempt at viral eradication using pegylated interferon plus ribavirin should be considered the first-line therapeutic option in patients with mild–moderate HCV-related MCS. Prolonged treatment (up to 72 weeks) may be considered in the case of virological non-responders showing clinical and laboratory improvements. Rituximab (RTX) should be considered in patients with severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis. High-dose pulsed glucocorticoid (GC) therapy is useful in severe conditions and, when necessary, can be considered in combination with RTX; on the contrary, the majority of conference participants discouraged the chronic use of low–medium GC doses. Apheresis remains the elective treatment for severe, life-threatening hyper-viscosity syndrome; its use should be limited to patients who do not respond to (or who are ineligible for) other treatments, and emergency situations. Cyclophosphamide can be considered in combination with apheresis, but the data supporting its use are scarce. Despite the limited available data, colchicine is used by many of the conference participants, particularly in patients with mild–moderate MCS refractory to other therapies. Careful monitoring of the side effects of each drug, and its effects on HCV replication and liver function tests is essential. A low-antigen-content diet can be considered as supportive treatment in all symptomatic MCS patients. Although there are no data from controlled trials, controlling pain should always be attempted by tailoring the treatment to individual patients on the basis of the guidelines used in other vasculitides. Conclusion Although there are few controlled randomised trials of MCS treatment, increasing knowledge of its pathogenesis is opening up new frontiers. The recommendations provided may be useful as provisional guidelines for the management of MCS.

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence ofBifidobacteriumin the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whetherBifidobacteriumwas able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species:Bifidobacterium lactis,B. bifidum, andB. longum. The binding of human plasminogen toBifidobacteriumwas dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strainB. lactisBI07. The data suggest that plasminogen binding toB. lactisis due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in theBifidobacterium-host interaction.

Probiotic bacteria play an important role in protecting the host from intestinal colonization of pathogenic bacteria. We have developed a new analytical approach based on a real-time PCR technique for quantifying Bifidobacterium adhesion to intestinal epithelial cells. Real-time PCR analysis showed that adhesion to enterocyte-like Caco-2 cells represented a variable phenotype in the genus Bifidobacterium, enabling classification of three adhesion behaviors: high adhesiveness (>40 bifidobacterial cells/Caco-2 cell); adhesiveness (5-40 bifidobacterial cells/Caco-2 cell); no adhesiveness (

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3–4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

Objective: To verify whether features of CNS involvement can be detected in SLE patients without overt neuropsychiatric manifestations.Methods: 114 SLE patients who had never received a diagnosis of neuropsychiatric lupus (never NPSLE) were studied and compared to 65 SLE patients with known neuropsychiatric involvement (NPSLE). The study relied on evaluation of neurocognitive functions by means of a battery of neuropsychological tests, on psychiatric and neuropsychological assessments and on neuroimaging studies (computed tomography, magnetic resonance, single photon emission computed tomography (SPECT)).Results: Clinical features, including disease duration/activity and pharmacological therapy, of never-NPSLE and NPSLE patients were similar. Short-term and long-term memory, visuo-spatial and verbal information processing were similarly compromised in never-NPSLE and in NPSLE patients; only attention was significantly more compromised in NPSLE patients. Psychiatric morbidity was higher than expected in never-NPSLE patients, although less than in the control neuropsychiatric group. Ischemic lesions, multiple small high intensity lesions and cortical atrophy, detected by CT and MR scans, as well as abnormal SPECT were also frequently detected in never NPSLE patients. Interestingly, left parietal and occipital area hypoperfusion by SPECT was significantly more frequent in the patients with impaired visuo-spatial intelligence and short-term memory.Conclusions: Most abnormalities detected by available diagnostic tools and characteristics of neuropsychiatric SLE are also present in non-symptomatic patients. They may derive from an unexpected widespread involvement of the CNS and are not per se sufficient, in the absence of clinical manifestations, for a diagnosis of neuropsychiatric SLE.

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)-mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Molecular cloning of the antibody repertoire in phage display combinatorial vectors is a powerful method enabling the dissection of the immunoresponse against a given pathogen. In this paper we describe the construction of a combinatorial library displayed on phage surface, containing the antibody repertoire of a patient with high serological response against hepatitis C virus (HCV) antigens. Following selection of the library against solid-phase-bound antigen, sixteen human antibody Fab fragments able to bind to HCV-specific antigens were generated and studied for binding characteristics. The majority of them appeared to have specificity for the HCV c33 peptide. All the clones reacting with the c33 peptide shared the same heavy-chain CDR3 sequence. This is the first report of molecular cloning in a combinatorial phage display vector of the antibody repertoire of an anti-HCV-positive patient.

Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in "pathobionts", i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing.

Objectives: Evaluation of the impact of a biscuit containing the probiotics Bifidobacterium longum Bar33 and Lactobacillus helveticus Barl3 on the intestinal microbiota in the elderly. Design: Randomized double-blind placebo-controlled trial. Participants: Thirty-two elderly volunteers living in Italy. The group was composed of 19 women and 13 men aged between 71 and 88 years (mean 76). Intervention: Subjects were randomized in two groups consuming one dose of the probiotics-containing biscuit or placebo once a day for 30 days. Measurements: For each subject the intestinal microbiota was characterized using the phylogenetic microarray platform HTF-Microbi. Array before and after intervention. Results: Our data demonstrated that one-month consumption of a probiotics-containing biscuit was effective in redressing some of the age-related dysbioses of the intestinal microbiota. In particular, the probiotic treatment reverted the age-related increase of the opportunistic pathogens Clostridium cluster XI, Clostridium difficile, Clostridium perfringens, Enterococcus faecium and the enteropathogenic genus Campylobacter. Conclusion: The present study opens the way to the development of elderly-tailored probiotic-based functional foods to counteract the age-related dysbioses of the intestinal microbiota. © 2013 Serdi and Springer-Verlag France.

SUMMARY. The aim of this study was to characterize hepatitis C virus (HCV) infection in patients with mixed cryoglobulinaemia (MC). The HCV RNA copy number was assayed in clinical specimens from 15 consecutive patients with MC and HCV infection. Absolute quantification of HCV RNA molecules was performed using a competitive reverse transcription-polymerase chain reaction (cRT-PCR). Specific HCV RNA sequences were detected and quantified in plasma samples from all patients (mean HCV RNA copy number 4.9 times 106 ml-1 plasma). A high concentration of HCV RNA molecules was detected in the cryoprecipitates of eight of the 15 patients, who had a cryoprecipitate/supernatant ratio higher than 3.0 (range 3.60 to 186.80); in the remain ing seven patients this ratio was close to or lower than 1.0 (range 0.13 to 1.60). Quantitative analysis of HCV RNA molecules in cells other than hepatocytes (i.e. peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs), in which the HCV replicative intermediate was detected using strand specific RT-PCR, demonstrated that infection is detectable in nearly 60% of these extrahepatic cells. Quantitative analysis of HCV RNA in PBMCs and BMCs revealed low levels of viral nucleic acids.

Human beings have been recently reviewed as ‘metaorganisms’ as a result of a close symbiotic relationship with the intestinal microbiota. This assumption imposes a more holistic view of the ageing process where dynamics of the interaction between environment, intestinal microbiota and host must be taken into consideration. Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbial ecosystem. Here we review the current knowledge of the changes occurring in the gut microbiota of old people, especially in the light of the most recent applications of the modern molecular characterisation techniques. The hypothetical involvement of the age-related gut microbiota unbalances in the inflamm-aging, and immunosenescence processes will also be discussed. Increasing evidence of the importance of the gut microbiota homeostasis for the host health has led to the consideration of medical/ nutritional applications of this knowledge through the development of probiotic and prebiotic preparations specific for the aged population. The results of the few intervention trials reporting the use of pro/ prebiotics in clinical conditions typical of the elderly will be critically reviewed.

Traditionally regarded as stable through the entire lifespan, the intestinal microbiota has now emerged as an extremely plastic entity, capable of being reconfigured in response to different environmental factors. In a mutualistic context, these microbiome fluctuations allow the host to rapidly adjust its metabolic and immunologic performances in response to environmental changes. Several circumstances can disturb this homeostatic equilibrium, inducing the intestinal microbiota to shift from a mutualistic configuration to a disease-associated profile. A mechanistic comprehension of the dynamics involved in this process is needed to deal more rationally with the role of the human intestinal microbiota in health and disease.

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumptionwere successfully screened, thus demonstrating that the developedmicroarray-based platformcould be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood. © 2011 Elsevier B.V. All rights reserved.

An emerging central concept in evolutionary biology suggests that symbiosis is a universal characteristic of living organisms that can help in understanding complex traits and phenotypes. During evolution, an integrative circuitry fundamental for survival has been established between commensal gut microbiota and host. On the basis of recent knowledge in worms, flies, and humans, an important role of the gut microbiota in aging and longevity is emerging. The complex bacterial community that populates the gut and that represents an evolutionary adapted ecosystem correlated with nutrition appears to limit the accumulation of pathobionts and infections in all taxa, being able of affecting the efficiency of the host immune system and exerting systemic metabolic effects. There is an urgent need to disentangle the underpinning molecular mechanisms, which could shed light on the basic mechanisms of aging in an ecological perspective. Thus, it appears possible to extend healthy aging and lifespan by targeting the host as a metaorganism by manipulating the complex symbiotic ecosystem of gut microbiota, as well as other possible ecosystems of the body.

In this review, we discuss the multifactorial role of intestinal microbiota in colorectal cancer. The peculiar metabolism of dietary compounds of the individual microbiota complement, its overall immunostimulation and immunomodulatory activity, and eventually the production of toxins that perturb the regulation of cell growth, define the balance of positive and negative risk factors for colorectal cancer development. Moreover, shaping the composition of the human intestinal microbiota, diet has an indirect impact in determining the balance between health and disease. The integration of diet, microbial, and host factors in a system approach is mandatory to determine the overall balance of risk and protective factors for colorectal cancer onset.

The intestinal bacterial ecosystem has gained increasing interest from the scientific community because of its demonstrated involvement in the aetiology and/or outcome of different physiopathological conditions, especially inflammatory diseases. The gut microbiota is affected by the aging process in terms of composition and functionality (Tiihonen et al., 2010). In a sort of self-sustaining loop, the aged-type microbiota has been hypothesized to contribute to several age-associated phenomena such as immunosenescence and inflammaging (Guigoz et al., 2008). The impact of the aging process on the gut microbiota has been addressed in several studies, some of which using modern molecular characterization techniques, thoroughly and carefully reviewed by Tihonen et al. (2010). However, an important contribution to the understanding of several aspects of this phenomenon has been provided by the two most recent studies in the field (Biagi et al., 2010; Claesson et al., 2010). These studies were performed with two different very up-to-date molecular techniques, the Human Intestinal Tract Chip (HITChip, a phylogenetic DNA microarray) and the pyrosequencing, which showed to be fairly comparable (Claesson et al., 2009; van de Bogert et al., 2011). The results described in the studies are quite unexpected and, taken together, can shed some light on very important question in the field.

Bifidobacteriumanimalissubsp.lactislives in the gastrointestinal tract of most mammals, including humans. Recently, for the probiotic strainB. animalissubsp.lactisBI07, a dose-dependent plasminogen-binding activity was demonstrated and five putative plasminogen-binding proteins were identified. Here we investigated the role of surface DnaK as aB. animalissubsp.lactisBI07 plasminogen receptor. DnaK was visualized on the bacterial cell surface by transmission electron microscopy. The His-tagged recombinant DnaK protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. The capability to tolerate physiological concentrations of bile salts is a crucial feature for an intestinal symbiont micro-organism. By proteome analysis we demonstrated that the long-term exposure ofB. animalissubsp.lactisBI07 to bile salts results in the upregulation of important surface plasminogen receptors such as DnaK and enolase. Moreover, adaptation ofB. animalissubsp.lactisBI07 to physiological concentrations of bile salts significantly increased its capacity to interact with the host plasminogen system. By enhancing the bacterial capacity to interact with the host plasminogen, the gut bile environment may facilitate the colonization of the human host byB. animalissubsp.lactisBI07.

Background Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation. Results In order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject. Conclusion The HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.

In this review we focus on the revision of the prebiotic concept in the context of the new metagenomic era. Functional metagenomic data provided by the Human Microbiome Project are revolutionizing the view of the symbiotic relationship between the intestinal microbiota and the human host. A deeper knowledge of the mechanisms that govern the dynamic interplay between diet, intestinal microbiota and host nutrition opens the way to better information on the prebiotic structure-function relationships, tailoring prebiotic formula into specific health attributes. On the other hand, functional genomic studies of the sourdough microbial communities allow to scan the environmental variability to identify novel metabolic traits for the biosynthesis of new potential prebiotic molecules. The integration of the functional analyses provided by the massive sequencing of bacterial genomes and metagenomes will allow the rational production of a desired prebiotic molecule with specific functional properties.

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

Rifaximin resistance in the probiotic strain Bifidobacterium infantis BI07 was studied to assess the use of an antibiotic-probiotic combination for clinical management of intestinal disorders. A rifaximin-resistant mutant was selected and a 129 bp core region of the rpoB gene was sequenced and compared with the respective sequence of the sensitive clone. A miss-sense mutation of codon 513, producing the substitution of Gln with Arg in the protein sequence, was found. The involvement of metabolic changes associated with rifaximin resistance was also investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. The principal categories of proteins, whose expression levels varied as a consequence of rifaximin resistance, included chaperonins, regulatory factors and metabolic enzymes. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was excluded, as neither structural modifications nor degradation derivates of the drug moiety was identified using liquid chromatography coupled with tandem mass spectrometry.

Methotrexate (MTX)-based chemotherapy extends survival in patients with primary brain lymphomas, but it is not clear whether multiagent chemotherapy is superior to MTX alone. Treatment options for patients with recurrent primary brain lymphoma are limited; there is no standard second-line chemotherapy. New chemotherapeutic agents with clear activity in brain lymphoma are needed for treatment of recurrent disease. We report the results of a phase II trial assessing activity of the alkylating agent temozolomide in immunocompetent patients with recurrent primary brain lymphomas, previously treated with high-dose MTX-containing chemotherapy and/or radiotherapy. A median of two courses (range 1-12) of temozolomide 150 mg m(-2) day(-1), for 5 days every 4 weeks was administered to 36 patients yielding nine complete and two partial responses (response rate: 31%; 95% confidence interval 16-46%). One-year survival was 31% ( 95% confidence interval 16-46%). Toxicity was negligible. We conclude that temozolomide is active in recurrent primary brain lymphomas and should further be evaluated in this disease, perhaps in combination with MTX as initial treatment.