Probiotics, prebiotics and synbiotics for weight loss and metabolic syndrome in the microbiome era.

OBJECTIVE: Excessive body fat and the associated dysmetabolic consequences affect both developed and emerging countries. An altered gut microbiota composition is an important environmental cause of these conditions. Clinical trials targeting gut microbiome composition or functions with pro or prebiotics to promote a healthier profile are considered a promising tool for excessive body weight treatment and prevention of dysmetabolic complications. MATERIALS AND METHODS: We searched PubMed and Cochrane Library using combinations of probiotics/prebiotics and synbiotics with obesity/weight loss/metabolic syndrome as the search terms. Clinical studies and significant pre-clinical results showing molecular mechanisms supporting clinical results were also discussed. RESULTS: Several studies in humans and in animal models have elucidated biological mechanisms supporting the observed clinical efficacy of selected probiotic and prebiotic compounds for weight management. Efficacy appears to be species or strain-specific. Fibers such as inulin or galactomannan promote independent and synergistic beneficial effects. CONCLUSIONS: Diet supplementation with synbiotics prepared using selected strains (such as Lactobacillus gasseri strains) showed to exert weight-reduction and anti-inflammatory activity in large independent studies. Their administration, together with galactomannan and/or inulin fibers, may increase weight management effects due to synergistic effect on short chain fatty acid production and microbiota 're-configuration'.

Targeting patients' microbiota with probiotics and natural fibers in adults and children with constipation.

OBJECTIVE: Functional constipation (FC) is a common condition in which the gut microbiota composition plays a fundamental role. The increasing knowledge on the role of gut microbes in the regulation of gut motility and stool consistency has allowed reconsidering, with a new scientific-based approach, the possibility to target the composition of intestinal bacterial populations for FC treatment. In this review, we evaluate recent attempts that used prebiotics, natural fibers or probiotics to treat FC, with a deep microbiome-based focus. MATERIALS AND METHODS: This is a literature review of articles published in Medline, Web of Science, and the Cochrane Library. Studies on FC in adults and children were identified using the following terms: constipation AND probiotics OR prebiotics OR synbiotics PR fibers OR microbiome OR microbiota. Selected animal studies were also considered if showing mechanistic observations. RESULTS: FC is associated with alteration in microbiome composition. Motility and fecal consistency are affected with different efficacy by the type of fiber, prebiotic or probiotic strain used in patients. CONCLUSIONS: Selected bacterial strains, mainly belonging to the Bifidobacterium genus, and some poorly or non-fermented natural fibers, such as Psyllium, may significantly improve FC and may represent the basis for an effective supplementation.

HIV DNA loads, plasma residual viraemia and risk of virological rebound in heavily treated, virologically suppressed HIV-infected patients.

In this single-centre, retrospective study, we analyzed data of 194 patients receiving antiretroviral therapy with <50 human immunodeficiency virus (HIV) RNA copies/mL in plasma and 318 HIV RNA/DNA paired samples. By kinetic polymerase chain reaction (kPCR) molecular system analysis, 104 (54%) subjects had undetectable HIV RNA and 90 (46%) had residual viraemia. Median (interquartile range) HIV DNA load was 780 (380-1930) copies/10(6) peripheral blood lymphocytes (PBL), and HIV DNA loads were independently associated with residual viraemia (p 0.002). Virological rebound occurred in 29/194 (15%) patients over a median (interquartile range) follow-up of 17.5 (13.5-31.5) months. Residual viraemia (p 0.002), but not HIV DNA load, was independently associated with virological rebound.

Virological rebound in human immunodeficiency virus-infected patients with or without residual viraemia: results from an extended follow-up.

Human immunodeficiency virus (HIV) -infected patients with HIV RNA loads of < 50 copies/mL were followed-up for a median (interquartile range) of 30.8 (11.7-32.9) months to study the effect of residual viraemia (RV) on virological rebound (VR). At baseline, 446 (60.3%) patients had undetectable HIV RNA (group A) and 293 (39.7%) had RV (1-49 HIV RNA copies/mL, group B) by kinetic PCR. VR occurred in 4 (0.9%) patients in group A and in 12 (4.1%) patients in group B (p 0.007). Time to VR was shorter among patients of group B (Log-rank test: p 0.003). However, the proportion of VR was extremely low also among patients with RV.

Evolution patterns of raltegravir-resistant mutations after integrase inhibitor interruption.

The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4-24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4(+) T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process.

Phage display for the production of human monoclonal antibodies against human pathogens.

In the last decade an increasing number of antibodies have made their way from the research benchtops into the clinics and many more are currently under clinical trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the molecular dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.

Intra-host evolution of human immunodeficiency virus type 1 and viral fitness.

RNA viruses are frequently tolerant to high levels of mutagenesis. In contrast, DNA viruses are less errorprone and coevolve along with their specific hosts over long time periods. Although both strategies have been successful, the "RNA-strategy" (directly linked to the pathogenic potential of these agents) most often generates novelty (new variants, new strains, and even new viral pathogens). For several decades, intra-host virus evolution has been considered to be a speculative field, far from the main issues of clinical virology. This concept is now changed, due to the evidence that RNA virus evolution is intimately linked to failures in viral disease control and prevention. Antiviral strategies using single and fixed elements (i.e. treatments using one antiviral compound, immunizations using a single recombinant protein) have been unable to control highly dynamic quasispecies, such as human immunodeficiency virus type I (HIV-1) and hepatitis C virus (HCV). The development of combinatorial treatments in HIV-1 infection and the recognition that vaccines should be multivalent are important steps in adapting disease control strategies to the complexity of viral populations. The present report summarizes the strategies adopted to address HIV-1 evolution and its phenotypic consequences, including changes in susceptibility to antiviral compounds, viral fitness, and pathogenic potential. In particular, it is highlighted that sequence-function analyses of the intra-host HIV-I evolution, including studies of viral fitness, have opened up new perspectives not only to studying the pathogenic mechanisms and the virus-host relationships, but also to designing new strategies for monitoring antiviral therapies.

Phylogenetic internal control for HIV-1 genotypic antiretroviral testing.

Genotypic testing includes several steps (RNA purification, RT-PCR amplification, DNA sequencing, sequence editing and analysis) that should be individually controlled. In our laboratory, we have added to this step-by-step internal control a final phylogenetic quality control: this is performed every time a sequence is obtained from a patient previously subjected to the same test. Each sequence with this characteristic is routinely compared with sequences from previous samples of the same patient by multiple alignment and a neighbor-joining tree by using Kimura two-parameter method is constructed. To validate the quality control procedure, we have aligned and calculated the mean similarity of the reverse transcriptase (first 984 nucleotides) and protease (whole gene) sequences from 30 patients whose virus was completely wild-type for both reverse transcriptase and protease. In the same tree, we have added the sequences obtained from 5 out of the 30 patients, tested at a second time point. The wild type sequences have shown a mean inter-sample divergence of 2.9%, and all the sequence pairs from individual patients clustered together in the tree constructed with the nucleotide sequences, while the tree constructed with the inferred aminoacid sequences did not always permit to cluster the sequences from the same patients. This indicates that: 1) the phylogenetic analysis of nucleic acid sequences can be useful to rule out sample mix-up; 2) the belonging of a sequence to each individual patient can efficiently be assessed also in the cases of extreme divergence in terms of drug resistance mutations.

Humoral immune response against hepatitis C virus.

Antibodies are in several instances a reliable marker indicating vigorous immune response against infectious agents and in several viral diseases presence in the blood of specific anti-viral antibodies indicates an effective protection. However, this is not always true. For example, in the case of hepatitis C virus (HCV) an important human pathogen considered the causative agent of the nonA- nonB hepatitis, in spite of an intense antibody response there is no protection against a new infection and in the majority of infected individuals the virus overcomes host defences establishing a persistent infection. Here we describe how the dissection of the humoral immune response against HCV glycoprotein E2 of infected patients was useful for a better comprehension of the virus-host interplay. Cross-reactive antibodies directed against E2 are produced by the HCV-infected patient, but not all of them are protective, and some could even result to be detrimental for the patient. The cross-reactive anti-HCV/E2 humoral antibody response is complex and not necessarily completely beneficial to the host.

Heterogeneity of the humoral anti-HCV/E2 response in persistently infected patients as demonstrated by divergent patterns of inhibition of the binding of anti-HCV/E2 human monoclonal antibodies.

A complete understanding of the molecular features of humoral immune response could be of pivotal importance in the management of persistent viruses as HCV. In this study, 24 HCV-positive samples, characterized by classical virological parameters, are evaluated using a new assay for the quantitation of antibody subpopulations directed against discrete epitopes on surface glycoprotein E2, a key viral protein. The results, besides confirming the usefulness of this new approach, highlight the extreme heterogeneity of anti-HCV/E2 response as far as single epitopes are concerned. The specific epitopes under study are also demonstrated to be widely shared among different genotypes.

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus.

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.

Characterization of GBV-C infection in HIV-1 infected patients.

BACKGROUND: GB virus C, a positive-stranded RNA virus, is classified in the family Flaviviridae. It is currently believed that persistent infection occurs in 25-50% of infected individuals, however, it still remains an "orphan" virus in search of a role in human pathology. Molecular epidemiological studies have demonstrated that GBV-C infection is present in about 1-1.4% of the healthy population in developed countries, that it shares routes of transmission with HIV and HCV and that the prevalence of GBV-C in these populations is higher than in blood donors. On the basis of the sequence variation among the isolates, GBV-C is classified into at least four major genotypes. Preliminary evidence has suggested that GBV-C is a lymphotropic virus that replicates mainly in the spleen and bone marrow. Recently, several reports have investigated the possible beneficial effect of GBV-C co-infection on HIV disease progression to AIDS, reduced mortality in HIV infected individuals and lower HIV viral loads, not leading to a definitive conclusion yet. AIM: To investigate the role of GBV virus C co-infection in two different subsets of HIV-infected patients, and to evaluate the prevalence of GBV-C genotypes in Northern Italy. METHODS: A total of 86 HIV positive patients were examined for GBV-C viremia (years after HIV sera conversion: 12 +/- 5). Control population (Group A): 46 patients (mean age 42 years) with <200CD4/ml during the observation period. Longterm non progressor population (Group B): 40 patients, (mean age 40 years) with >500 CD4/ml for at least 8 years and never treated with HAART. After extraction of viral RNA from plasma samples, amplification of a highly conserved region of 5'UTR was performed by nested RT-PCR. All positive samples were genotyped by sequencing, alignment with published sequences and phylogenetic analysis. CD4 cell count, HIV plasma levels were also evaluated. RESULTS: 9 out of 46 (19.56%) in Group A and 15 out of 40 (37.5%) in Group B had detectable GBV-C viremia (p=0.064, OR 2.47, percent confidence interval 0.94 to 6.51). No statistical difference was observed when disease stage was evaluated between the two groups. In Group B, after regression analysis for CD4 cell count decrease over the period observed, no significant difference was detected between GBV-C positive and negative patients. No significant difference was observed in Group B in HIV viremia and CD4 cell count at time of GBV-C detection between GBV-C infected patients and GBV-C negative patients. All Italian patients were genotype 2, the only African patient carried GBV-C genotype 1. CONCLUSIONS: Although previous results suggest that GBV-C virus may be a favorable marker for long term non progression of HIV disease, whether it plays a direct anti-HIV role or just takes advantage of non progessors' higher CD4 cell count to replicate more efficiently, still remains to be answered. Follow up of untreated patients and further evaluation of virological interactions, between the viruses and the host immune system, will be helpful to shed some light on these observations, offering new prognostic and eventually therapeutical tools for the management of HIV patients.

Probiotics and Helicobacter pylori eradication.

The need for new strategies for Helicobacter pylori eradication, alternative or complementary to antibiotic therapy, has recently claimed the attention of many investigators. Pre-clinical studies have shown the inhibition of Helicobacter pylori growth by Lactobacilli and the anti-Helicobacter pylori action of Lactobacillus salivarius, Lactobacillus acidophilus and Lactobacillus casei subspecies rhamnosus strains, possibly due to the production of lactic acid or to the secretion of an autolysin. Clinical studies have demonstrated a persistent reduction in delta over baseline values at the 13C urea breath test independently of omeprazole administration with Lactobacillus acidophilus La1, the eradication in 6 out of 14 patients with Lactobacillus acidophilus alone, positive results in patients in which a standard Helicobacter pylori triple therapy was randomly supplemented with Lactobacillus acidophilus.

Helicobacter pylori treatment: a role for probiotics?

Many new therapeutic strategies are studied to improve Helicobacter pylori eradication rate. Probiotics are live microorganisms which, upon administration, may interact with the human microflora and positively affect the health status. The use of probiotics in the field of H. pylori infection has been proposed for improving eradication rate and tolerability and for compliance of multiple antibiotic regimens used for the infection. Results from laboratory studies and from clinical trials seem to confirm the expectancies, but there is lack of standardization in terms of type of probiotic strain used, dosage and timing of supplementation. Before further ongoing trials and future studies will clarify these points, probiotics could remain a useful adjunct to standard anti-H. pylori therapies, but cannot take the place of other validated options.

Effect of Lactobacillus GG supplementation on antibiotic-associated gastrointestinal side effects during Helicobacter pylori eradication therapy: a pilot study.

BACKGROUND: One-week triple therapy is currently regarded as the reference of anti-Helicobacter pylori treatment. However, antibiotic-associated gastrointestinal side effects are among the major pitfalls of such regimens. Probiotic supplementation may be regarded as a therapeutic tool to prevent or reduce these troublesome drug-related manifestations. AIM: To determine whether the addition of the probiotic Lactobacillus GG to an anti-H. pylori standard triple therapy could help to prevent or minimize the occurrence of gastrointestinal side effects. METHODS: One hundred and twenty healthy asymptomatic subjects screened positive for H. pylori infection and deciding to receive eradication therapy were randomized either to 1-week pantoprazole (40 mg b.i.d.), clarithromycin (500 mg b.i.d.), tinidazole (500 mg b.i.d.) or to the same regimen supplemented with Lactobacillus GG for 14 days. Patients filled in validated questionnaires during follow-up to determine the type and severity of side effects and to judge overall tolerability. RESULTS: Bloating, diarrhea and taste disturbances were the most frequent side effects during the eradication week and were significantly reduced in the Lactobacillus GG-supplemented group (RR = 0.4, CI 0.2-0.8; RR = 0.3, CI 0.1-0.8; RR = 0.3, CI 0.1-0.7, respectively). The same pattern was observed throughout the follow-up period. Overall assessment of treatment tolerability showed a significant trend in favor of the Lactobacillus GG-supplemented group (p = 0.03). CONCLUSIONS: Lactobacillus GG supplementation beneficially affects H. pylori therapy-related side effects and overall treatment tolerance.

The effect of oral administration of Lactobacillus GG on antibiotic-associated gastrointestinal side-effects during Helicobacter pylori eradication therapy.

BACKGROUND: One-week triple therapy is currently considered the golden standard against Helicobacter pylori. However, gastrointestinal side-effects are among the major pitfalls in such regimens. Probiotic supplementation might help to prevent or reduce such drug-related manifestations. AIM: To determine whether adding the probiotic Lactobacillus GG to an anti-H. pylori regimen could help to prevent or minimize the gastrointestinal side-effects burden. METHODS: Sixty healthy asymptomatic subjects screened positive for H. pylori infection were randomized to 1 week rabeprazole (20 mg b.d.), clarithromycin (500 mg b.d.), tinidazole (500 b.d.) and the probiotic Lactobacillus GG for 14 days or to the same regimen with a placebo preparation. Patients completed validated questionnaires during the week of treatment and during the following 3 weeks, to determine the type and severity of side-effects and an overall judgement of tolerability. RESULTS: Diarrhoea, nausea and taste disturbance were significantly reduced in the Lactobacillus GG supplemented group (relative risk=0.1, 95% CI: 0.1-0.9; relative risk=0.3, 95% CI: 0.1-0.9; relative risk=0.5, 95% CI: 0.2-0.9, respectively). An overall assessment of treatment tolerability showed a significant difference in favour of the Lactobacillus GG supplemented group (P=0.04). CONCLUSIONS: Lactobacillus GG supplementation showed a positive impact on H. pylori therapy-related side-effects and on overall treatment tolerability.

A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates.

BACKGROUND: Acid suppression plus two antibiotics is considered the reference anti-Helicobacter pylori treatment. Reported eradication rates are around 65-80%. Human Lactobacillus acidophilus shows an in vitro inhibitory effect on the attachment of H. pylori to gastric epithelial cell lines. Culture supernatant of this bacillus seems to decrease the in vitro viability of H. pylori. AIM: To evaluate whether the supplementation with an inactivated preparation of L. acidophilus could improve the efficacy of a standard anti-H. pylori therapy. METHODS: One-hundred and twenty H. pylori-positive patients were randomly assigned to a 7-day triple therapy based on rabeprazole (20 mg b.d.), clarithromycin (250 mg t.d.s.) and amoxicillin (500 mg t.d.s.) (RCA group: 60 subjects), or to the same regimen supplemented with a lyophilized and inactivated culture of Lactobacillus acidophilus (t.d.s.) (RCAL group: 60 subjects). RESULTS: In the RCA group, eradication was successful in 72% (42 out of 58 patients) from a per protocol (PP) analysis, or 70% (42 out of 60 patients) using an intention-to treat (ITT) analysis. In the RCAL group a significant increase in the eradication rate was observed: 88% (52 out of 59 patients) from PP analysis (P=0.03), 87% (52 out of 60 patients) from ITT analysis (P=0.02). CONCLUSIONS: These results seem to confirm the in vitro anti-H. pylori effect of L. acidophilus, suggesting that the inactivated L. acidophilus could be effective in increasing eradication rates of a standard anti-H. pylori therapy.

Efficacy of a multistep strategy for Helicobacter pylori eradication.

BACKGROUND: Helicobacter pylori eradication therapies do not achieve 100% success rates. Antibiotic resistant strains are among the major causes of failure. Current recommendations concerning the management of treatment failures are not fully clear. AIM: To evaluate the efficacy of a multi-step therapeutic strategy in a large group of infected patients. METHODS: A total of 2606 H. pylori-positive patients were administered tinidazole, clarithromycin and a proton pump inhibitor for 1 week. Patients with continuing infection were then given a second 1-week course of amoxycillin, clarithromycin and ranitidine bismuth citrate. Patients still infected after the second course underwent upper gastrointestinal endoscopy with H. pylori culture, and then received a 1-week quadruple proton pump inhibitor-bismuth based scheme established on H. pylori antibiotic sensitivity. RESULTS: After the first step, eradication was achieved in 2063 out of 2413 patients [86% per protocol analysis (PP); 79% intention-to-treat analysis (ITT)]. First-step failures (350 out of 2413; 14.5% PP) showed second-step eradication rates of 82% (271 out of 329 patients, PP; 77% ITT). The specific quadruple therapy for second-step failures (58 out of 329, 18% PP) achieved 77% (30 out of 39 patients, PP) or 52% (ITT) success. This algorithm led to overall eradication rates of 99% (PP) or 91% (ITT). CONCLUSIONS: This multi-step strategy succeeded in a high percentage of H. pylori infected patients. Given the lack of precise guidelines on treatment failures, assessing H. pylori sensitivity to antibiotics only after failure of the second treatment could be suggested in clinical practice.