Diego Saita

Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV)

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

Oral Probiotic VSL#3 Prevents Autoimmune Diabetes by Modulating Microbiota and Promoting Indoleamine 2,3-Dioxygenase-Enriched Tolerogenic Intestinal Environment

The gut microbiota modulates the autoimmune pathogenesis of type 1 diabetes (T1D) via mechanisms that remain largely unknown. The inflammasome components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis. In this study we show that modifications of the gut microbiota induced by oral treatment with Lactobacillaceae-enriched probiotic VSL#3, alone or in combination with retinoic acid (RA), protect NOD mice from T1D by affecting inflammasome at the intestinal level. In particular, we show that VSL#3 treatment inhibits IL-1β expression while enhancing release of protolerogenic components of the inflammasome, such as indoleamine 2,3-dioxygenase (IDO) and IL-33. Those modifications of the intestinal microenvironment in VSL#3-treated NOD mice modulate gut immunity by promoting differentiation of tolerogenic CD103+ DCs and reducing differentiation/expansion of Th1 and Th17 cells in the intestinal mucosa and at the sites of autoimmunity, that is, within the pancreatic lymph nodes (PLN) of VSL#3-treated NOD mice. Our data provide a link between dietary factors, microbiota composition, intestinal inflammation, and immune homeostasis in autoimmune diabetes and could pave the way for new therapeutic approaches aimed at changing the intestinal microenvironment with probiotics to counterregulate autoimmunity and prevent T1D.

In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir

OBJECTIVES The cross-resistance profiles of elvitegravir and dolutegravir on raltegravir-resistant variants is still controversial or not available in macrophages and lack extensive evaluations on wide panels of clonal variants. Thus, a complete evaluation in parallel with all currently available integrase inhibitors (INIs) was performed. METHODS The integrase coding region was RT-PCR-amplified from patient-derived plasma samples and cloned into an HIV-1 molecular clone lacking the integrase region. Twenty recombinant viruses bearing mutations to all primary pathways of resistance to raltegravir were phenotypically evaluated with each integrase inhibitor in freshly purified CD4+ T cells or monocyte-derived macrophages. RESULTS Y143R single mutants conferred a higher level of raltegravir resistance in macrophages [fold change (FC) 47.7-60.24] compared with CD4+ T cells (FC 9.55-11.56). All other combinations had similar effects on viral susceptibility to raltegravir in both cell types. Elvitegravir displayed a similar behaviour both in lymphocytes and macrophages with all the tested patterns. When compared with raltegravir, none to modest increases in resistance were observed for the Y143R/C pathways. Dolutegravir maintained its activity and cross-resistance profile in macrophages. Only Q148H/R variants had a reduced level of susceptibility (FC 5.48-18.64). No variations were observed for the Y143R/C (+/-T97A) or N155H variants. CONCLUSIONS All INIs showed comparable antiretroviral activity in both cell types even if single mutations were associated with a different level of susceptibility in vitro to raltegravir and elvitegravir in macrophages. In particular, dolutegravir was capable of inhibiting with similar potency infection of raltegravir-resistant variants with Y143 or N155 pathways in both HIV-1 major cell reservoirs.

Antigen-Driven Evolution of B Lymphocytes in Coronary Atherosclerotic Plaques

Recent data indicated that adaptive immunity is involved in the process of atherogenesis. Oligoclonal recruitment of T lymphocytes has been described in coronary plaques of patients with acute coronary syndrome. However, the nature of immune response remains to be determined. In the present study, we examined the Ab response in six coronary plaques obtained by endoluminal directional atherectomy. The IgG1/κ-coding gene repertoires of B lymphocytes present in circulating blood and in coronary plaques were cloned and analyzed. In all of the six plaques, we observed 1) a skewed usage of heavy and light IgG1/κ Ab-coding genes, 2) an oligoclonal distribution of VK, JK, and VH, DH, and JH genes with overrepresentation of some rarely used IgG genes, and 3) the unequivocal signs of Ag-driven clonal expansion and evolution of B cells. The data document for the first time the presence of a local Ag-driven clonal evolution of B cells in human atherosclerotic plaques.

Duodenal mucosa of patients with type 1 diabetes shows distinctive inflammatory profile and microbiota

Context Increasing evidences suggest a correlation between gut and type 1 diabetes (T1D). Objective The objective of this study is to evaluate the gut inflammatory profile and microbiota in patients with T1D compared with healthy control (CTRL) subjects and patients with celiac disease (CD) as gut inflammatory disease controls. Design/Setting/Participants The inflammatory status and microbiome composition were evaluated in biopsies of the duodenal mucosa of patients with T1D (n = 19), in patients with CD (n = 19), and CTRL subjects (n = 16) recruited at San Raffaele Scientific Institute, in Milan, Italy, between 2009 and 2015. Main Outcome Measures Inflammation was evaluated by gene expression study and immunohistochemistry. Microbiome composition was analyzed by 16S ribosomal RNA gene sequencing. Results An increased expression of CCL13, CCL19, CCL22, CCR2, COX2, IL4R, CD68, PTX3, TNFα, and VEGFA was observed in patients with T1D compared with CTRL subjects and patients with CD. Immunohistochemical analysis confirmed T1D-specific inflammatory status compared with healthy and CD control tissues, mainly characterized by the increase of the monocyte/macrophage lineage infiltration. The T1D duodenal mucosal microbiome results were different from the other groups, with an increase in Firmicutes and Firmicutes/Bacteroidetes ratio and a reduction in Proteobacteria and Bacteroidetes. The expression of genes specific for T1D inflammation was associated with the abundance of specific bacteria in the duodenum. Conclusions This study shows that duodenal mucosa in T1D presents disease-specific abnormalities in the inflammatory profile and microbiota. Understanding the mechanisms underlying these features is critical to disentangle the complex pathogenesis of T1D and to gain new perspectives for future therapies targeting the intestine.

The Microbiome of the Prostate Tumor Microenvironment

BACKGROUND The advent of molecular-based methods of identification and characterization of complex microbial populations has led to a new era of microbial discovery. A detailed and comprehensive analysis of the microbial ecosystem of the pathologic and healthy prostate tissues has not been yet reported. OBJECTIVES To characterize the microbiome possibly associated to the pathologic prostate microenvironment. DESIGN, SETTING, AND PARTICIPANTS The microbiome profile of tumor, peri-tumor, and nontumor tissues was assessed on 16 radical prostatectomy-specimens. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Microbiome analysis was assessed by massive ultradeep pyrosequencing. Bacteria load was expressed as a percentage of the total number of bacteria. The statistical significance of differences among specimen-groups was tested with Friedmans test (Dunn posthoc test) and Wilcoxon rank-sum test. RESULTS AND LIMITATIONS Three phyla, six classes, nine orders, 14 families, and 11 genera were above the set threshold value of 1%, respectively. Significant differences in specific microbial populations among tumor/peri-tumor and nontumor prostate specimens were observed at certain taxonomic levels. Among genera, Propionibacterium spp. were the most abundant. Staphylococcus spp. were more represented in the tumor/peri-tumor tissues (p<0.05). The restricted number of specimens represents a potential limitation. CONCLUSIONS The prostate contains a plethora of bacteria, which set themselves within the gland with a distribution dependent on the nature of the tissue, thus suggesting a possible pathophysiological correlation between the composition of the local microbial niche and the presence of the tumor itself. Future studies will help to clarify the role of these specific bacteria and their potential to be exploited as new biomarkers. PATIENT SUMMARY The pathological prostate is populated by specific microbial populations, whose distribution varies according to the nature of the tissue. This finding opens interesting perspectives for the identification of novel therapeutic approaches and biomarkers.

Adaptive immunity against gut microbiota enhances apoE-mediated immune regulation and reduces atherosclerosis and western-diet-related inflammation

Common features of immune-metabolic and inflammatory diseases such as metabolic syndrome, diabetes, obesity and cardiovascular diseases are an altered gut microbiota composition and a systemic pro-inflammatory state. We demonstrate that active immunization against the outer membrane protein of bacteria present in the gut enhances local and systemic immune control via apoE-mediated immune-modulation. Reduction of western-diet-associated inflammation was obtained for more than eighteen weeks after immunization. Immunized mice had reduced serum cytokine levels, reduced insulin and fasting glucose concentrations; and gene expression in both liver and visceral adipose tissue confirmed a reduced inflammatory steady-state after immunization. Moreover, both gut and atherosclerotic plaques of immunized mice showed reduced inflammatory cells and an increased M2 macrophage fraction. These results suggest that adaptive responses directed against microbes present in our microbiota have systemic beneficial consequences and demonstrate the key role of apoE in this mechanism that could be exploited to treat immune-metabolic diseases.

Cross-Reacting Antibacterial Auto-Antibodies Are Produced within Coronary Atherosclerotic Plaques of Acute Coronary Syndrome Patients

Coronary atherosclerosis, the main condition predisposing to acute myocardial infarction, has an inflammatory component caused by stimuli that are yet unknown. We molecularly investigated the nature of the immune response within human coronary lesion in four coronary plaques obtained by endoluminal atherectomy from four patients. We constructed phage-display libraries containing the IgG1/kappa antibody fragments produced by B-lymphocytes present in each plaque. By immunoaffinity, we selected from these libraries a monoclonal antibody, arbitrarily named Fab7816, able to react both with coronary and carotid atherosclerotic tissue samples. We also demonstrated by confocal microscopy that this monoclonal antibody recognized human transgelin type 1, a cytoskeleton protein involved in atherogenesis, and that it co-localized with fibrocyte-like cells transgelin+, CD68+, CD45+ in human sections of coronary and carotid plaques. In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions. Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota). From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin. These findings demonstrated that in human atherosclerotic plaques a local cross-reactive immune response takes place.

2-Aminothiazolones as Anti-HIV Agents That Act as gp120-CD4 Inhibitors

ABSTRACT We report here the synthesis of 2-aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors.

The new and less toxic protease inhibitor saquinavir–NO maintains anti-HIV-1 properties in vitro indistinguishable from those of the parental compound saquinavir

Although, the antiviral activity, tolerability and convenience of protease inhibitors have improved significantly in recent years, toxicity-associated adverse events including diarrhea, lipid alterations, disturbance of glucose homeostasis and liver enzyme elevations still remain a major concern during treatment of HIV-1 patients. We have recently shown that the covalent attachment of the NO moiety to the HIV-1 protease inhibitor saquinavir (Saq-NO) reduces its toxicity. In this study, we evaluated in vitro the anti-HIV activity of Saq-NO vs. its parental compound Saq. Site directed mutants with the most frequently identified Saq associated resistance mutations and their combinations were generated on proviral AD8-based backbones. Phenotypic assays were conducted using wild type clinical isolates and fully replicating recombinant viruses with Saq and Saq-NO in parallel on purified CD4+ T cells. The following recombinant viruses were generated and tested: L33F, M46I, G48V, I54V, I84V + L90M, M46I + L90M, G48V + L90M, M46I + I54V + L90M, L33F + M46I + L90M. The fold change resistance compared to the wild type viruses was between 1.3 and 7 for all single mutants, between 3.4 and 20 for double mutants and between 16.7 and 28.5 for viruses carrying three mutations for both compounds. The results clearly demonstrate that Saq-NO maintains an anti-HIV-1 profile very similar to that of Saq. The possibility to reduce Saq associated side effects and to increase the concentration of the drug in vivo may allow a higher and possibly more effective dosage of Saq-NO in HIV-1-infected patients and to increase the genetic barrier of this PI thus impairing the selection of resistant clones.