Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine

Abnormal Liver Blood Tests in Patients with Hyperthyroidism: Systematic Review and Meta-Analysis

Background: Abnormal liver blood tests (LBTs) in hyperthyroid patients are not uncommonly encountered. One major adverse event of antithyroid drug (ATD) therapy is drug-induced hepatotoxicity. Abnormal LBT in the hyperthyroidism scenario is a main diagnostic and therapeutic dilemma. We aimed to assess the prevalence and the response to ATD therapy of LBT abnormalities in newly diagnosed and uncomplicated hyperthyroidism through a systematic review and meta-analysis. Methods: A literature search was performed reporting LBTs at presentation and after ATD therapy in hyperthyroid patients. A proportion meta-analysis was performed with random-effects model. Pooled data were presented with 95% confidence intervals (CI). I2 statistic index was used to quantify the heterogeneity. Sensitivity analyses for prevalence of hyperthyroid patients with at least one abnormal LBT were performed. p-Value of <0.05 was regarded as significant. Results: The literature search yielded 2286 studies, of which 25 were included for systematic review and meta-analysis. The prevalence of untreated hyperthyroid and Graves' disease patients with at least one abnormal LBT was 55% ([CI 46-63%], I2 96%) and 60% ([CI 53-67%], I2 92%), respectively. The prevalence of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (BIL), and γ-glutamyltransferase (GGT) abnormalities in hyperthyroid patients were 33% ([CI 24-44%], I2 95%), 23% ([CI 17-29%], I2 89%), 44% ([CI 35-52%], I2 93%), 12% ([CI 7-20%], I2 92%), and 24% ([CI 16-36%], I2 95%), respectively. ATD therapy, along with euthyroidism restoration, was accompanied by normalization of LBT abnormalities in the following percentage of cases: ALT 83% ([CI 72-90%], I2 46%), AST 87% ([CI 74-94%], I2 2%), ALP 53% ([CI 32-73%], I2 76%), BIL 50% (CI cannot be calculated), and GGT 70% ([CI 47-87%], I2 74%). The sensitivity analyses showed similar results as those of the main analyses. The publication bias was not statistically significant for all outcomes, except for the prevalence of resolved BIL abnormalities that was not calculable. Conclusions: LBT abnormalities are common in newly diagnosed and untreated hyperthyroidism setting. A high chance of safely normalizing elevated transaminases, up to fivefold above the upper limit of normal, accompanies the use of ATDs in the treatment of hyperthyroidism

Preclinical carotid atherosclerosis enhances the global cardiovascular risk and increases the rate of cerebro- and cardiovascular events in a five-year follow-up

AIM: To evaluate if the intima-media thickening (IMT) and asymptomatic carotid plaque (ACP), as expression of carotid preclinical atherosclerosis (pre-ATS), can provide further information on the global cardiovascular risk (GCVR). METHODS: We studied 454 asymptomatic subjects, with a cluster of risk factors (RF), and evaluated the incidence of a first cardiovascular (CV) event in a five-year follow-up. The subjects at admission were subdivided in three groups of risk. RESULTS: Events occurred in 38% of subjects at high risk, in 13% and 6% of subjects at intermediate and low risk (p<0.003). Among evaluated parameters, carotid pre-ATS was a predictive marker of CV events (OR 2.7, 95% IC 1.4-5.1, p<0.0024). In subjects with GCVR<20% the prevalence of events was 8% for normal carotid ultrasound findings, 13% for increased IMT and 15% for ACP. CONCLUSIONS: In primary prevention, the IMT measurement can give further information for a better stratification of GCVR. The pre-ATS of carotid arteries should be considered a strong predictor of future CV events and should suggest a more aggressive treatment of RF

Change in circulating levels of endothelial progenitor cells and sexual function in women with type 1 diabetes

Context: Endothelial progenitor cells (EPCs), which are involved in the mechanisms of vascular repair, and sexual function are decreased in diabetic women as compared with general population. Objective: To investigate the circulating levels of EPCs and the change in sexual function during the menstrual cycle in women with type 1 diabetes (T1DM) compared with healthy women. Design: Case-control observational study. Setting: Unit of Endocrinology and Metabolic Diseases at University Hospital "Luigi Vanvitelli'' of Naples. Patients: Thirty-six women with T1DM and 64 age-matched healthy controls. Intervention: EPCs were quantified by flow cytometry and sexual function was assessed using the Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale. All the assessments were made at the follicular, ovulatory and luteal phases of the same menstrual cycle. Main outcome measures: Differences in EPCs levels and sexual function between patients and controls. Results: Compared with controls, women with diabetes showed significantly lower levels of both CD34+ (P &lt; 0.001) and CD34+CD133+ cells (P &lt; 0.001) in the ovulatory phase, and CD34+KDR+ cells in both the ovulatory phase and in the luteal phase (P &lt; 0.001 for both). Diabetic women showed significantly lower total FSFI scores and higher FSDS score than control women in all the phases of the menstrual cycle. FSFI total score was predicted by both CD34+CD133+ and CD34+KDR+ cells in the follicular phase, CD34+ and CD34+KDR+CD133+ cells in the ovulatory phase, CD34+KDR+ and CD34+KDR+CD133+ cells in the luteal phase. Conclusion: Women with T1DM show lower levels of EPCs during the menstrual cycle, compared with controls. EPCs count predict sexual function in this selected population

Analysis of the Streptococcus agalactiae exoproteome

International audienceThe two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates

Immunobiological characterization of genetic products, identified from whole genome lambda-display libraries of Pneumococcus

Objectives: Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide, including meningitis. Here, we employed the isogenic Δspr0075-, Δspr1370- and Δspr1875-knock-out mutants of the DP1004 pneumococcal strain to investigate the role of these proteins in the interaction with microglia, the resident brain macrophages. Methods: By screening a whole genome phage display library with sera from patients, we identified clones carrying pneumococcal B-cell epitopes. Among these, we isolated six antigenic fragments of sequences matching three previously unidentified proteins, encoded by the ORFs spr0075, spr1370 and spr1875 of R6 strain genomic sequence. By using an in vitro infection model and a gentamicin protection assay we evaluated, respectively, the ability of microglial BV2 cells to phagocytose and kill the isogenic mutants and the survival of these bacteria inside the microglia. Results: All strains were efficiently internalized by BV2 cells; yet the levels of phagocytosis obtained with Δspr0075 strain were lower than those observed with the other groups. The survival of the deleted strains inside the microglia was significantly different. The residual CFU of Δspr1370 and Δspr1875 mutants were, indeed, respectively, higher and lower, than those observed with parental DP1004. Moreover, preliminary results indicate that a similar trend is also observed in a bactericidal assay using BV2 as effector cells. Conclusion: These findings indicate that the proteins encoded by spr1370 and spr1875 loci are involved in interaction between S. pneumoniae and microglia

Binding of mAb K20 to <i>Candida albicans</i> cell wall, particularly on germ tubes and budding cells.

<p>MAb K20 labeled with biotin was used in the immunofluorescence assay with streptavidin-fluorescein.</p

Binding of phage clones to mAb K20.

<p>Plates were sensitized with a mAb directed against phage protein III (1 µg/ml) and 100 µl of purified phage clones (10¹¹ pfu/ml) were added. After 1 h incubation, mAb K20 or an isotype-matched IgM (1 µg/ml) were added. Binding was detected by using alkaline phosphatase-conjugated anti-mouse IgM Ab. Pools of libraries, (pVIII-12aa/pVIII.Cys-Cys or pVIII-9aa.Cys/pVIII.Cys-Cys or pVIII-9aa/pVIII-9aa.Cys) were used in different phage selections. Results are reported in separate panels (A, B or C). Phage pC89, displaying wild-Type pVIII, was used as negative control. Data represents the means ± the SD of three determinations.</p

Aminoacid sequences of phage inserts from mAb K20-binding clones.

<p>*peptide libraries of 12 (pVIII-12aa and pVIII.Cys-Cys) or 9 (pVIII-9aa) amino acids.</p><p>Aminoacid sequences of phage inserts from mAb K20-binding clones.</p