KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi

Background

Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE) with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi.

Methods

Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA) were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge.

Results

Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4⁺CD62L^lowCCR7^low effector memory T cells pre- and post-sand fly challenge.

Conclusions

This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection.     

Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics.

Methods

X- ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied.

Results

We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p.Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1–12.7) and 24.7 (19.8–29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation.

Conclusions

This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.     

Systemic and mucosal immune responses to sublingual or intramuscular human papilloma virus antigens in healthy female volunteers

The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19-31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12) or intramuscular injection (IM, n = 6). IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ~38-fold lower serum and ~2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans. TRIAL REGISTRATION: ClinicalTrials.govNCT00949572.     

Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.     

Oxidation of dibenzothiophene (DBT) by Serratia marcescens UCP 1549 formed biphenyl as final product

ABSTRACT: BACKGROUND: The desulphurization of dibenzothiophene (DBT), a recalcitrant thiophenic fossil fuel component by Serratia marcescens (UCP 1549) in order for reducing the sulphur content was investigated. The study was carried out establishing the growth profile using Luria Bertani medium to different concentrations of DBT during 120hours at 28oC, and orbital shaker at 150rpm. RESULTS: The results indicated that concentrations of DBT 0.5, 1.0 and 2.0 mM do not affected the growth of the bacterium. The DBT showed similar Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MCB) (3.68 mM). The desulphurization of DBT by S. marcescens was used with 96 hours of growth on 2mM of DBT, and was determined by gas chromatography (GC) and GC-mass spectrometry. In order to study the desulphurization process by S. marcescens was observed the presence of a sulfur-free product at 16 hours of cultivation The results show that S. marcescens oxidizes DBT to its corresponding DBT-5 oxide and then to DBT-sulfone, without the formation of any biphenyl. CONCLUSIONS: The data suggests the use of metabolic pathway "4S" by S. marcescens (UCP 1549) and formed biphenyl. The microbial desulphurization process by S. Serratia can be suggest significant reducing sulphur content in DBT, and showed promising potential for reduction of the sulfur content in diesel oil.     

Rapid virus dissemination in infant macaques after oral simian immunodeficiency virus exposure in the presence of local innate immune responses

A vaccine to protect human immunodeficiency virus (HIV)-exposed infants is an important goal in the global fight against the HIV pandemic. Two major challenges in pediatric HIV vaccine design are the competence of the neonatal/infant immune system in comparison to the adult immune system and the frequent exposure to HIV via breast-feeding. Based on the hypothesis that an effective vaccine needs to elicit antiviral immune responses directly at the site of virus entry, the pattern of virus dissemination in relation to host immune responses was determined in mucosal and lymphoid tissues of infant macaques at 1 week after multiple oral exposures to simian immunodeficiency virus (SIV). The results show that SIV disseminates systemically by 1 week. Infant macaques can respond rapidly to virus challenge and mount strong innate immune responses. However, despite systemic infection, these responses are most pronounced in tissues close to the viral entry site, with the tonsil being the primary site of virus replication and induction of immune responses. Thus, distinct anatomic compartments are characterized by unique cytokine gene expression patterns. Importantly, the early response at mucosal entry sites is dominated by the induction of proinflammatory cytokines, while cytokines with direct antiviral activity, alpha/beta interferons, are only minimally induced. In contrast, both antiviral and proinflammatory cytokines are induced in lymphoid tissues. Thus, although infant macaques can respond quickly to oral viral challenge, the locally elicited immune responses at mucosal entry sites are likely to favor immune activation and thereby virus replication and are insufficient to limit virus replication and dissemination.     

Complementary adhesin function in C. albicans biofilm formation

BACKGROUND: Biofilms are surface-associated microbial communities with significant environmental and medical impact. Here, we focus on an adherence mechanism that permits biofilm formation by Candida albicans, the major invasive fungal pathogen of humans. RESULTS: The Als surface-protein family has been implicated in biofilm formation, and we show that Als1 and Als3 have critical but redundant roles. Overexpression of several other Als proteins permits biofilm formation in a biofilm-defective als1/als1 als3/als3 strain, thus arguing that the function of Als proteins in this process is governed by their respective expression levels. The surface protein Hwp1 is also required for biofilm formation, and we find that a mixture of biofilm-defective hwp1/hwp1 and als1/als1 als3/als3 strains can form a hybrid biofilm both in vitro and in vivo in a catheter infection model. Complementary function of Hwp1 and Als1 and 3 seems to reflect their interaction because expression of Hwp1 in the heterologous host S. cerevisiae permits adherence to wild-type C. albicans, but not to an als1/als1 als3/als3 strain. CONCLUSIONS: The complementary roles of Hwp1 and Als1 and Als3 in biofilm formation are analogous to the roles of sexual agglutinins in mating reactions. This analogy suggests that biofilm-adhesin complementarity may promote formation of monospecies biofilms.     

A porphyrin‐based fluorescence method for zinc determination in commercial propolis extracts without sample pretreatment

The quantification of zinc in over‐the–counter drugs as commercial propolis extracts by molecular fluorescence technique using meso ‐tetrakis(4‐carboxyphenyl)porphyrin (H₂TCPP⁴) was developed for the first time. The calibration curve is linear from 6.60 to 100 nmol L^?1 of Zn²⁺. The detection and quantification limits were 6.22 nmol L^?1 and 19.0 nmol L^?1, respectively. The reproducibility and repeatability calculated as the percentage variation of slopes of seven calibration curves were 6.75% and 4.61%, respectively. Commercial propolis extract samples from four Brazilian states were analyzed and the results (0.329–0.797 mg/100 mL) obtained with this method are in good agreement with that obtained with the Atomic Absorption Spectroscopy (AAS) technique. The method is simple, fast, of low cost and allows the analysis of the samples without pretreatment. Moreover the major advantage is that Zn‐porphyrin complex presents fluorescent characteristic promoting the selectivity and sensitivity of the method.