Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein I

Background: The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. Results: Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT. Conclusion: An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore.

Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study.

OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.

Development and standardization of solid phase assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). A report on the second phase of an international cooperative study on the standardization of ANCA assays

Anti-neutrophil cytoplasmic antibodies (ANCA) are diagnostic markers for systemic vasculitis. They are classically I detected by an indirect immunofluorescence test using normal donor neutrophils as substrate. This assay lacks antigenic specificity and is not quantitative. The 'EC/BCR Project for ANCA Assay Standardization' is an international collaboration study with the aim to develop and standardize solid phase assays for ANCA detection. In this part of the study the isolation and characterization of proteinase-3 and myeloperoxidase, the two main target molecules for ANCA, and the development and standardization of ELISAs with these antigens are described. Six laboratories successfully isolated purified proteinase-3 preparations that could be used. Three of these preparations, together with one myeloperoxidase preparation, were subsequently used for ANCA testing by ELISA. The ELISA technique was standardized in two rounds of testing in the 14 participating laboratories. The coefficient of variation of these new assays decreased from values of approx. 50% in the first round to approx. 20% in the second round. We conclude that purified proteinase-3 and myeloperoxidase can be used in standardized ELISAs for ANCA detection. Whether such procedures offer advantages over the IIF test will be determined in a prospective clinical study.

A study of HPV 1, 2 and 4 antibody prevalence in patients presenting for treatment with cutaneous warts to general practitioners in N. Ireland

Three hundred and seventy-six patients attending their general practitioner with cutaneous warts at five health centres in Northern Ireland were screened for human papilloma virus (HPV) types 1 and 2 IgM antibody using an indirect immunofluorescence test. Eight-eight (23.4%) patients were positive for HPV type 1 IgM and 156 (41.5%) for HPV type 2 IgM. HPV 1 IgM antibody was significantly more likely to be associated with plantar warts than warts elsewhere (P less than 0.0001). HPV 2 IgM was present in 45 (34.1%) patients with plantar warts and 99 (45.6%) patients with warts at other sites (P = 0.1). Evidence of multiple infection by HPV types 1 and 2 was demonstrated by the finding of HPV 1 and 2 IgM antibodies in the sera of 16 (4.3%). HPV 4 was found in only 1 out of 30 biopsies and HPV 4 IgM was undetectable in 50 randomly chosen sera.

Leukocyte entry into the CNS of Leishmania chagasi naturally infected dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Soroprevalência da infecção por Trypanosoma cruzi em cães de uma área rural do Estado de Mato Grosso do Sul

Doença de Chagas é uma antropozoonose causada por Trypanosoma cruzi que tem os cães como importante reservatório da doença na América do Sul. Este trabalho teve como objetivo avaliar a ocorrência da infecção natural pelo T. cruzi em cães de uma área rural do estado de Mato Grosso do Sul, Brasil. Foram utilizados os testes de imunofluorescência indireta (IFI) e ensaio imunossorvente ligado a enzima (ELISA) em 75 cães residentes na área. Foram detectados anticorpos em 45,3% (n=34) e 24,0% (n=18) nos testes de IFI e ELISA, respectivamente. A real prevalência da infecção foi confirmada como 22,7% (n=17) pelo critério de positividade em ambos os testes. Os resultados obtidos confirmam a infecção chagásica nos cães dessa região.

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell and humoral immunity in endemic pemphigus foliaceus

Foram avaliados dezesseis doentes portadores de pênfigo foliáceo endêmico, dez com a forma localizada da doença (Grupo G1) e seis com a forma disseminada (Grupo G2), com os objetivos de correlacionar o quadro clínico e laboratorial desses pacientes com o perfil imunológico dos mesmos, e verificar a relação dos títulos dos anticorpos antiepiderme circulantes, identificados pela imunofluorescência indireta, com intensidade da lesão e com a evolução das lesões em tratamento. Foram realizados: hemograma completo, quantificação de subpopulação de células mononucleares por anticorpos monoclonais e estudo da transformação blástica de linfócitos e quantificação de anticorpos circulantes por meio da reação de imunofluorescência indireta. Observou-se leucocitose principalmente no grupo G2, diminuição dos valores relativos das subpopulações de linfócitos CD3+ e CD4+ e tendência à diminuição dos valores relativos da subpopulação CD8+ nos doentes (Grupos G1 e G2). Os índices de transformação blástica de linfócitos frente à fitohemaglutinina revelaram níveis mais elevados nos doentes (Grupos G1 + G2), que nos controles. A reação de imunofluorescência indireta foi positiva em 100% dos doentes do grupo G2 e em 80% do grupo G1 A mediana dos valores dos títulos foi maior no grupo G2, quando comparado com o grupo G1. A análise global dos resultados permite concluir que a imunidade celular está preservada, e que existe uma relação entre os títulos de anticorpos obtidos à reação de imunofluorescência indireta e extensão da lesão cutânea.

Renal involvement in canine leishmaniasis: a morphological and immunohistochemical study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CLINICAL STUDY of NATURAL INFECTION BY Trypanosoma cruzi IN DOGS RESIDING IN A RURAL AREA IN MATO GROSSO do SUL STATE, BRAZIL

This study was carried out to describe the clinical characteristics of natural infection caused by Trypanosoma cruzi in dogs that reside in a rural area of Mato Grosso do Sul State, Brazil. Conventional and nonconventional diagnostic methods were used for screening T. cruzi infection in 75 dogs that lived in the area. Cardiovascular tests and biochemical examination of sera were also performed in four confirmed positive dogs. The following techniques were employed: indirect immunofluorescence test (IFAT), enzyme-linked immunosorbent assay with T. cruzi epimastigote antigens (EAE-ELISA) and enzyme-linked immunosorbent assay with T. cruzi excreted-secreted trypomastigote antigens (TESA-ELISA) with antibodies detected in 45.33% (n = 34), 24.0% (n = 18) and 12.0% (n = 9) of the dogs, respectively. The current prevalence of the infection was confirmed as 10.7% (n = 8) by immunoblotting test with T. cruzi excreted-secreted antigens (TESA-blot). The test that showed the best concordance index (Kappa; 0.93), sensitivity (100%) and specificity (98.5%) was TESA-ELISA, that when associated with IFAT had the same results as those obtained by TESA-blot (10.7%). Three out of the four chagasic animals showed enlarged cardiac silhouette on X-ray and an increase of the P-wave duration and QRS complex in electrocardiogram. Two dogs presented conduction disturbances, right bundle branch block in one dog and first-degree atrioventricular block and sinus arrest in another. The ecodopplercardiography presented left-ventricular-wall thickness increased during diastole, decrease of the shortening fraction and inversion in the speed peaks of the E and A waves, indicating the presence of systolic and diastolic disorders. The four animals showed enzymatic activities of creatine kinase (221-404 U/L), MB fraction of creatine kinase (189-304 U/L), elevated total proteins (7.6-10.2 g/dL) and total globulins (4.6-7.7g/dL) and reduction of albumin/globulin ratio, which suggested a myocardial injury and continuous antigenic stimulus.

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the São Manuel abattoir, São Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%). © 2001 Elsevier Science B.V.

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm. © 2003 Elsevier Ltd. All rights reserved.

Soroprevalência de Toxoplasma gondii em rebanhos caprinos no Estado de São Paulo

Four hundred forty-two serum samples were collected from dairy goats in seven regions of São Paulo State. These were tested for Toxoplasma gondii antibodies using the indirect immunofluorescent antibody test. Sixty-four (14,5%) serologically positive animals were found from all these goat farms studied.

Diferenciação hepatocítica de células-tronco mesenquimais da medula óssea humana

Some recent articles have reported that mesenchymal stem cells (MSCs) can be induced to express hepatocyte markers by transplanting them into animal models of liver damage, or by in vitro culture with growth factors and cytokines. In this study, the aim is to evaluate the behavior of MSCs subjected to induction of hepatocyte differentiation. The MSCs were isolated from the bone marrow of 4 normal donors, characterized and subjected to both in vitro and in vivo induction of hepatocyte differentiation. The in vitro induced cells showed morphological changes, acquiring hepatocyte-like features. However, the immunophenotype of these cells was not modified. The induced cells exhibited no increase in albumin, cytokeratin 18 or cytokeratin 19 transcripts, when analyzed by real-time RT-PCR. The expression of albumin, cytokeratin 18 and alpha fetoprotein was also unchanged, according to immunofluorescence tests. In vivo, the MSC demonstrated a potential to migrate to damaged liver tissue in immunodeficient mice. Taken together, the results suggest that bone marrow MSCs are incapable of in vitro differentiation into hepatocytes by the approach used here, but are capable of homing to damaged hepatic tissue in vivo, suggesting a role for them in the repair of the liver. This contribution to tissue repair could be associated with a paracrine effect exerted by these cells.