Assessment of potters' occupational exposure to lead and associated risk factors in Maragogipinho, Brazil: preliminary results.

BACKGROUND AND PURPOSE: Lead (Pb) is used as a flux in the glazing process of pottery utensils in several regions of the world. It can affect the nervous and hematopoietic systems; in addition, it is classified as a probable human carcinogen. This work aims to evaluate Pb exposure of potters and describe the main determinants of elevated blood Pb (PbB) levels in this group of workers. METHODS: This is a cross-sectional study with potters of Maragogipinho Village, Bahia, Brazil, of both sexes, aged 16-72 years (n = 85). Non-exposed workers of the same age range residing in the urban area of Aratuípe town were also recruited (n = 50). We evaluated Pb dust deposition rates (PbDrt) in pottery workshops and PbB levels. All Pb measurements were performed by Graphite Furnace Atomic Absorption Spectrometry. RESULTS: The median of PbB (min-max) and geometric mean (SD) PbDrt for the exposed group were 7.9 (0.9-49.8) µg/dL and 1463 (± 290,000) µg/m2/30 days, respectively. For the control group, levels were 1.5 (0.1-19.8) µg/dL and 82 (46) µg/m2 30 days, respectively. CONCLUSION: The data found showed an excessive exposure among artisans, exceeding occupationally safe levels and those reported in the literature. It is important to implement occupational hygiene measures and improvements of the working conditions of these labors, especially the replacement of lead oxide in the pottery-glazing process.

Leishmania eukaryotic elongation Factor-1 beta protein is immunogenic and induces parasitological protection in mice against Leishmania infantum infection.

Treatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.

Zinc-protoporphyrin determination by HPLC with fluorescence detection as a biomarker of lead effect in artisanal pottery workers.

Lead (Pb) exposure compromises heme synthesis by inhibiting ferrochelatase, forming zinc-protoporphyrin (ZnPP). This study aims to validate a method for the determination of ZnPP by HPLC with fluorescence detection and apply this method to evaluate the extent of Pb exposure of artisanal pottery workers. Extraction procedures were tested using both nonacid and acid liquid-liquid extraction. The former presented a better chromatogram and recovery results. The validated method yielded a good resolution of ZnPP and its free form peaks with acceptable precision and accuracy. Total run time was 15 min and ZnPP peak retention time was 5.6 min. We applied this method to evaluate 39 potters (90% male), mean age 40 years (9-80). The medians (ranges) of blood lead, ZnPP and hemoglobin were 16.0 µg/dl (2.2-71.5), 12.6 µg/dl (4.6-279.8) and 15.1 g/dl (11.0-17.8), respectively. Significant differences were observed for blood lead according to gender, age range (>40 years), direct handling of lead oxide and years of occupation. Significantly higher levels of ZnPP were observed in male potters involved with lead glazing activity. The validated method was shown to be simple with one-step nonacid extraction, good sensitivity, reproducibility and accuracy. Our data shows that these laborers are dangerously exposed to Pb, reflecting the effect on the heme synthesis.

Recombinant Leishmania eukaryotic elongation factor-1 beta protein: A potential diagnostic antigen to detect tegumentary and visceral leishmaniasis in dogs and humans.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

TipMT: Identification of PCR-based taxon-specific markers.

BACKGROUND: Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. RESULTS: Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. CONCLUSIONS: The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .

Corneal Elevation Topography in Primary Open Angle Glaucoma.

PURPOSE OF THE STUDY: The purpose of the study was to describe and compare anterior and posterior topographic elevation maps in primary open angle glaucoma patients with functional damage staging and in healthy controls. METHODS: A total of 217 subjects were consecutively recruited, including 111 primary open angle glaucoma patients (patients), and 106 healthy individuals (controls). All patients performed Pentacam HR corneal topography. Mean anterior keratometry and anterior and posterior topographic elevation maps were compared in the central 3, 5, and 7 mm. Humphrey automated perimetry results from the patient group were classified according to the Glaucoma Staging System. RESULTS: Age (patients: 72.32±8.09; controls: 70.82±8.36; P=0.18) and central corneal pachymetry (patients: 541.13±36.98; controls: 548.67±34.56; P=0.12) were similar in both groups. Maximum elevation readings in the central 5 mm were significantly (P<0.05) higher in the anterior (patients: 8.21±8.63; controls: 5.79±3.62) and posterior (patients: 16.17±8.72; controls: 13.92±6.03) corneal topography of the glaucomatous patients, as well as in the anterior (patients: 17.32±20.78; controls: 9.61±5.64) and posterior (patients: 38.81±19.78; controls: 26.38±12.73) central 7 mm. There was a weak but significant correlation between the Glaucoma Staging System stage and both the anterior 5 mm (r=0.397) and 7 mm (r=0.304) maximum, as well as the posterior 5 mm (r=0.233) and 7 mm (r=0.241) maximum. CONCLUSIONS: In patients with primary open angle glaucoma, there is a forward shifting of the posterior and anterior corneal surfaces. This appears to be correlated with more advanced stages of functional damage, pointing to a possible link between corneal structural changes and duration and intensity of elevated intraocular pressure. Further studies may ascertain the potential for this biological marker to be used in monitoring primary open angle glaucoma patients.

CYP1B1 gene analysis and phenotypic correlation in Portuguese children with primary congenital glaucoma.

PURPOSE: To investigate the prevalence of CYP1B1 mutations in Portuguese children with primary congenital glaucoma (PCG) and to study the possible correlations between the mutation status and clinical features of the disease. METHODS: DNA sequencing analysis of the CYP1B1 gene was used to screen 21 children with PCG followed on Paediatric Ophthalmology and Medical Genetics consultations at D. Estefânia's Hospital (Centro Hospitalar de Lisboa Central, Portugal). The effect of mutations on the phenotype of the patients was also assessed. Presence and type of mutations in CYP1B1 gene, age at diagnosis, bilaterality, age at first surgery, postoperative intraocular pressure and corneal diameter, final visual acuity, number of surgical reinterventions, and number of antiglaucoma medications required postoperatively were noted. RESULTS: Mutations in the CYP1B1 gene in 6 patients (28.57%) were detected, all compound heterozygotes. Seven types of mutations were identified: c.182G>A, c.317C>A, c.535delG, c.1064_1076del, c.1159G>A, c.1310C>T, and c.1390dupT. All patients with these mutations developed bilateral PCG, whereas in the group without mutations only 7 (46.67%) showed bilateral disease. Age at diagnosis was lower in the group of patients with these mutations (0.0 ± 0.00 vs 4.5 ± 2.63 months, p<0.01). In the remaining variables (age at first surgery, postoperative intraocular pressure and corneal diameter, final visual acuity, number of surgical reinterventions and antiglaucoma medications required postoperatively), no significant differences between the groups were detected (p>0.05 for all comparisons). CONCLUSIONS: This study is the first to report the variety of mutations in the CYP1B1 gene in a group of Portuguese children with PCG and to describe 2 new mutations. Genetic analysis of PCG must be carried out, although it has not yet been possible to establish a genotype-phenotype correlation, with the exception of bilaterality and early age at diagnosis.

Evasion of the Immune Response by Trypanosoma cruzi during Acute Infection.

Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people mainly in Latin America. To establish a life-long infection, T. cruzi must subvert the vertebrate host's immune system, using strategies that can be traced to the parasite's life cycle. Once inside the vertebrate host, metacyclic trypomastigotes rapidly invade a wide variety of nucleated host cells in a membrane-bound compartment known as the parasitophorous vacuole, which fuses to lysosomes, originating the phagolysosome. In this compartment, the parasite relies on a complex network of antioxidant enzymes to shield itself from lysosomal oxygen and nitrogen reactive species. Lysosomal acidification of the parasitophorous vacuole is an important factor that allows trypomastigote escape from the extremely oxidative environment of the phagolysosome to the cytoplasm, where it differentiates into amastigote forms. In the cytosol of infected macrophages, oxidative stress instead of being detrimental to the parasite, favors amastigote burden, which then differentiates into bloodstream trypomastigotes. Trypomastigotes released in the bloodstream upon the rupture of the host cell membrane express surface molecules, such as calreticulin and GP160 proteins, which disrupt initial and key components of the complement pathway, while others such as glycosylphosphatidylinositol-mucins stimulate immunoregulatory receptors, delaying the progression of a protective immune response. After an immunologically silent entry at the early phase of infection, T. cruzi elicits polyclonal B cell activation, hypergammaglobulinemia, and unspecific anti-T. cruzi antibodies, which are inefficient in controlling the infection. Additionally, the coexpression of several related, but not identical, epitopes derived from trypomastigote surface proteins delays the generation of T. cruzi-specific neutralizing antibodies. Later in the infection, the establishment of an anti-T. cruzi CD8(+) immune response focused on the parasite's immunodominant epitopes controls parasitemia and tissue infection, but fails to completely eliminate the parasite. This outcome is not detrimental to the parasite, as it reduces host mortality and maintains the parasite infectivity toward the insect vectors.

Identification of secreted virulence factors of Chromobacterium violaceum.

Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.

Identification and functional analysis of Trypanosoma cruzi genes that encode proteins of the glycosylphosphatidylinositol biosynthetic pathway.

BACKGROUND: Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. CONCLUSIONS/SIGNIFICANCE: Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.