Chiara Valsecchi

In vitro activation of mononuclear cells by two probiotics: Lactobacillus paracasei I 1688, Lactobacillus salivarius I 1794, and their mixture (PSMIX).

Background: Most studies on probiotics have described their effects on the human immune system after ingestion of LAB, but little is known about their effect on in vitro stimulation of human immune cells. Aim of the study: Evaluate the “in vitro” activity of Lactobacillus paracasei (I 1688), Lactobacillus salivarius (I 1794), and a commercial mix of the two (PSMIX, Proge Farm), on immune cells from healthy individuals. Materials: Two probiotic strains, Lactobacillus salivarius (I 1794; Proge Farm, Italy) and Lactobacillus paracasei (I 1688; Proge Farm, Italy), which are contained in the functional food ENTEROBACILLI, were evaluated for their ability to stimulate peripheral blood mononuclear cells and modulate surface phenotype and cytokine production. Results: All subjects responded to the bacteria, with different levels of response. The cell populations that showed a significant percent increase were CD4+/CD25+ cells (T-helper activated regulatory cells), CD8+/CD25+ (T-suppressor/cytotoxic activated cells), and CD16+/CD56+ (NK cells) (p<0.05). IL-12 and IFN-γ in vitro production significantly increased with exposure to probiotics (p<0.05 for both). Conclusions: This study provides the first evidence that Lactobacillus paracasei and Lactobacillus salivarius are capable of inducing a specific immune response that may be useful in the clinical setting for improving innate and adaptive immune responses.

Probiotics and food allergy.

The exact prevalence of food allergy in the general population is unknown, but almost 12% of pediatric population refers a suspicion of food allergy. IgE mediated reactions to food are actually the best-characterized types of allergy, and they might be particularly harmful especially in children. According to the “hygiene hypothesis” low or no exposure to exogenous antigens in early life may increase the risk of allergic diseases by both delaying the development of the immune tolerance and limiting the Th2/Th1 switch. The critical role of intestinal microbiota in the development of immune tolerance improved recently the interest on probiotics, prebiotics, antioxidants, polyunsaturated fatty acid, folate and vitamins, which seem to have positive effects on the immune functions.Probiotics consist in bacteria or yeast, able to re-colonize and restore microflora symbiosis in intestinal tract. One of the most important characteristics of probiotics is their safety for human health. Thanks to their ability to adhere to intestinal epithelial cells and to modulate and stabilize the composition of gut microflora, probiotics bacteria may play an important role in the regulation of intestinal and systemic immunity. They actually seem capable of restoring the intestinal microbic equilibrium and modulating the activation of immune cells.Several studies have been recently conducted on the role of probiotics in preventing and/or treating allergic disorders, but the results are often quite contradictory, probably because of the heterogeneity of strains, the duration of therapy and the doses administered to patients. Therefore, new studies are needed in order to clarify the functions and the utility of probiotics in food allergies and ion other types of allergic disorders.

From IgE to clinical trials of allergic rhinitis.

The current scientific research is continuously aiming at identifying new therapeutic targets with the purpose of modifying the immune response to allergens. The evolution in immunological methods has led to the identification of immunoglobulin E (IgE) as both a diagnostic biomarker and potential therapeutic target in allergic diseases, such as allergic rhinitis. Allergen immunotherapy has been used for more than 100 years to treat allergic diseases and it is today considered the only disease-modifying treatment capable of inducing a long-lasting immunological and clinical tolerance toward the causal allergen. During the past 20 years, major advances have been made in understanding the molecular and cellular mechanisms of allergen tolerance in humans. Moreover, there has been considerable progress in allergen extract modifications and additions to standard extracts. The recognition that IgE plays a pivotal role in basic regulatory mechanisms of allergic inflammation has recently stimulated research into the therapeutic potential of directly targeting this antibody. Omalizumab, the most advanced humanized anti-IgE monoclonal antibody, is currently approved for the treatment of uncontrolled allergic asthma and chronic spontaneous urticaria. Interesting results also arise from studies in which omalizumab was administered in patients with allergic rhinitis. The aim of this review is to provide an update on current findings on immunological and clinical effects of allergen immunotherapy and anti-IgE therapy, which have been shown to have synergistic modes of action for the treatment of allergic rhinitis.

Passive exposure to smoke results in defective interferon-γ production by adenoids in children with recurrent respiratory infections.

There is evidence that exposure to passive smoke is associated with an increased susceptibility to respiratory infections. Indeed, cigarette smoke extracts may interfere with the immune system, even though the precise mechanism has not been fully understood yet. Recurrent respiratory infections may be sustained by a defective immune response. The aim of the present study was to evaluate whether, in a cohort of children presenting both with recurrent respiratory infections and with a history of exposure to tobacco smoke, these factors were related to a lower local production of interferon-gamma (IFN-gamma) when compared to a similar non-exposed population. The study group included 128 children undergoing adenoidectomy, presenting with more than three respiratory infections per year, independently of exposure to passive smoke at home. The intracellular cytokine profile of lymphocyte subsets in adenoids was evaluated by flow cytometry analysis. Children exposed to tobacco smoke suffered from a significantly greater number of respiratory infections and had a lower percentage of IFN-gamma-producing CD8+ cells in adenoids than non-exposed children, while other T-cell subsets were not affected. The effect of smoke exposure seems to be specific to the IFN-gamma-producing CD8+ cells in adenoids and may contribute to the increased susceptibility to the recurrence of respiratory infections.

B lymphocyte subsets and their functional activity in the early months of life

In the present study we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated “natural antibody” production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p<0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p<0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

Effect of Lactobacillus paracasei CNCM I-1572 on symptoms, gut microbiota, short chain fatty acids, and immune activation in patients with irritable bowel syndrome: A pilot randomized clinical trial:

Background Evidence suggests a role of intestinal microbiota-host interactions in the pathophysiology and symptoms of irritable bowel syndrome (IBS). Objective The objective of this article is to assess the effects of Lactobacillus paracasei CNCM I-1572 on clinical and gut microbiota-related factors in IBS. Methods We conducted a multicenter, randomized, double-blind, cross-over, 18-week, placebo-controlled, pilot trial assessing the effect of Lactobacillus paracasei CNCM I-1572 on symptoms, gut microbiota composition, fecal short chain fatty acid (SCFA), immunoglobulin A, and cytokines in IBS. The intestinal microbial ecosystem was characterized by 16S rRNA gene profiling. Results Forty IBS patients were enrolled from five Italian centers. Lactobacillus paracasei CNCM I-1572 did not significantly improve IBS symptoms, including primary efficacy variables worst abdominal pain/discomfort and IBS degree of relief. Interestingly, Lactobacillus paracasei CNCM I-1572 induced a significant reduction in genus Ruminococcus, dominated by taxa related to Ruminococcus bromii and Ruminococcus callidus, a significant increase in the SCFAs acetate and butyrate, and a significant reduction in the pro-inflammatory cytokine interleukin-15. Conclusions This pilot study shows that Lactobacillus paracasei CNCM I-1572 is able to modulate gut microbiota structure/function and reduce immune activation in IBS. As no statistically significant effect on IBS-symptoms was found, further studies are necessary to determine the role of this probiotic in IBS. The study was registered at ClinicalTrials.gov registry under identifier NCT02371499.

Probiotics and Vaccination in Children

Immunisation is one of the most beneficial and cost-effective disease prevention measures. However several immunisations are associated with suboptimal seroconversion rates and so the protective effect is not optimal. In the last two decades the concept about the use of probiotic bacteria as novel mucosal adjuvants has engendered a lot of interest due to our increased immunological understanding and the availability of various techniques to enhance existing vaccine specific-immune responses. Mostly in developing countries, many people still die every year from vaccine-preventable diseases such as pneumonia and diarrhea. To date, emphasis has been placed on identifying novel vaccine antigens and adjuvants that induce stronger protective immune responses, as well as developing mucosally-administered vaccines. We would have enormous benefits in allowing safe administration of vaccines in remote areas and we may overcome the necessity for multiple doses. The precise mechanism of action of probiotics is not fully understood, but several animal and human studies have proven immunomodulatory effects involving both the humoral and cellular components of the host’s immune system. This review discusses whether dietary supplementation with oral probiotics enhances the immune response of infants after routine vaccinations and also evaluates clinical effects of probiotics in adults. Further well designed, randomized, placebo-controlled studies are needed to understand fully the immunomodulatory properties of probiotics, whether the effects exerted are strain and age-dependent, and their clinical relevance in enhancing protection following vaccination.

Gut Microbiota and Obesity.

Intestinal microbiota is composed by symbiotic innocuous bacteria and potential pathogens also called pathobionts. The human gut normally hosts roughly 1014 bacterial organisms of up to 1000 different species. The genome size of this microbial organ, collectively named microbiome, exceeds the size of the human nuclear genome by 2 orders of magnitude.

Impact of child obesity on adipose tissue physiology: assessment of adipocytokines and inflammatory cytokines as biomarkers of obesity

Obesity could be interpreted as a low grade inflammatory state. The role of cytokines for innate and acquired immune response and adipocytokines in pathogenesis of obesity is not completely understood. The aim of the study was to evaluate anthropometric parameters, adipocytokines and inflammatory cytokine levels as biomarkers of childhood obesity. This investigation was designed as a longitudinal observational study. Forty-seven obese children (19 males and 28 females) were enrolled by Pediatric Clinic of the Foundation IRCCS Policlinico San Matteo, Pavia, Italy. For each patients a blood sample, used for other biochemical evaluations, was collected. Cytokines and adipocytokines plasmatic levels were determined using an ELISA method. Plasma leptin levels are in correlation with age (r=0.5; P<0.001) and BMI-z score (r=0.36; P<0.001), particularly in girls; plasma resistin levels are in inverse correlation with age, particularly in boys (r=-0.67; P<0.001) and in correlation with BMI-z score (r=0.52; P=0.002). Plasma leptin and resistin levels show a good correlation with antrophometric parameters of child obesity (sex and BMI z score). This study suggests that leptin and resistin can be considered as biomarker of childhood obesity and its comorbility. We observed a statistically significant correlation between plasma leptin and resistin levels and antrophometric parameters of child obesity (sex and BMI z score). This study suggests that adipocytokines, such as leptin and resistin, can be considered as biomarkers of childhood obesity.

Impact of passive smoke and/or atopy on adenoid immunoglobulin production in children.

The adenoids are exposed to a wide number and variety of microbes, environmental pollutants, and food antigens. Atopy and passive smoke may significantly affect immune responses, mainly in children. The aim of the present study was to investigate whether passive exposure to tobacco smoke and/or atopy could affect immunoglobulin production by adenoidal lymphocytes in a cohort of children presenting with adenoid hypertrophy. A total of 277 children (151 males and 126 females; median age 5.5 years), with adenoidal hypertrophy requiring adenoidectomy and or adeno-tonsillectomy, were consecutively enrolled in the study. Adenoid mononuclear cells were in vitro stimulated with LPS or CpG. When considering both the presence of smoke exposure and atopy, we observed that the CpG-induced decrease in IgA and IgM production was significantly associated with this combination of risk factors. In the T-independent immunoglobulin production assay we found a positive association between the two risk factors and IgA and IgM production. In particular, the presence of both risk factors, showed a significant increase in IgA and IgM production after stimulation. In conclusion, this is the first study that investigated the in vitro adenoidal B cell response after different stimuli in children, also evaluating possible exposure to passive smoke and/or an atopic condition.