Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm) utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H) através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.

Frequência de anticorpos contra o vírus da língua azul em ovinos do estado do Ceará, Brasil

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

Aspectos clínico-laboratoriais da infecção natural por Trypanosoma cruzi em cães de Mato Grosso do Sul

A doença de Chagas causada pelo T. cruzi é transmitida, principalmente, por insetos vetores e está distribuída na Argentina, no Chile, na Venezuela e no Brasil. O cão, além de ser um importante reservatório, também é vítima da doença e a única espécie capaz de desenvolver manifestações clínicas iguais a do homem. O presente trabalho teve como objetivo descrever as alterações clínicas encontradas em quatro cães infectados naturalmente pelo T. cruzi e alertar para a possibilidade de que a ocorrência dessa enfermidade em cães de Mato Grosso do Sul possa estar sendo subestimada. Os animais foram selecionados a partir de exames sorológicos de reação de imunofluorescência indireta (RIFI), ensaio imunossorvente ligado à enzima (ELISA) e immunoblotting com antígeno secretado e excretado da forma tripomastigota do T. cruzi (TESA-blot) e submetidos a xenodiagnóstico, exame físico, radiografia torácica, eletrocardiografia, ecocardiografia e bioquímica sérica. As alterações encontradas foram aumento de ventrículo direito, presença de arritmias do tipo bloqueio átrio ventricular, sinus arrest e bloqueio de ramo direito, além de disfunção sistólica e diastólica. Três animais apresentaram hiperproteinemia e as dosagens das enzimas CK e CK-MB revelaram valores indicativos de uma miocardite ativa. Esses são os primeiros casos descritos de cães com evidências consistentes de infecção natural pelo T. cruzi em Mato Grosso do Sul e ressalta-se o alerta aos médicos veterinários para a importância clínica e o papel dessa espécie como reservatório da doença.

BINDING OF PARACOCCIDIOIDES-BRASILIENSIS TO LAMININ THROUGH SURFACE GLYCOPROTEIN GP43 LEADS TO ENHANCEMENT OF FUNGAL PATHOGENESIS

Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a K-d of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil

A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlandia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N, caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1 %) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T: gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By TP-Nc, two highly immunodominant antigens (29 and 35 kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N, caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Glucose- and insulin-induced phosphorylation of the insulin receptor and its primary substrates IRS-1 and IRS-2 in rat pancreatic islets

The presence of tyrosine-phosphorylated proteins was studied in cultured rat pancreatic islets, Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM glucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insulin led to a substantial increase in the phosphorylation of all three bands, with no appearance of novel bands. Immunoprecipitation with specific antibodies demonstrated that the signal detected at 95 kDa corresponds to the beta subunit of the insulin receptor (IR) while the band at 165-185 kDa corresponds to the early substrates of the insulin receptor, IRS-1 and IRS-2. Immunoprecipitation with IRS-I or IRS-2 antisera detected their association with the lipid metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), Thus, this is the first demonstration that elements involved in the insulin-signalling pathway of traditional target tissues are also present in pancreatic islets and are potentially involved in auto- and paracrine-signalling in this organ.

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: Optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiveroperating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P-neg=0.83), but a poor positive concordance (P-pos=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninuin was significantly associated with T gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis. (c) 2006 Elsevier B.V. All rights reserved.

Detection of immunoglobulin G antibodies to Neospora caninum in humans: High seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders

Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.

Hypoactivity of the central dopaminergic system and autistic-like behavior induced by a single early prenatal exposure to lipopolysaccharide

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.

Detection of the 43,000-molecular-weight glycoprotein in sera of patients with paracoccidioidomycosis

The 43,000-molecular-weight (43K) soluble glycoprotein was detected in sera of patients with paracoccidioidomycosis by the immunoblot technique by using as the probe rabbit monospecific antisera to this fraction. The 43K antigen was present before treatment in sera of patients with the acute (juvenile) form; it started to disappear from circulation after 10 months of chemotherapy, and it was undetectable afer 2 years of treatment. In the chronic cases, the 43K antigen was detected in patients without treatment, and it was absent in the healed cases. The detection of the 43K protein specific to Paracoccidioides brasiliensis may be important for its diagnostic value as well as for modulation of the host immune response.

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

ANALISE ANTIGENICA DE PROTEASES DE DIFERENTES CEPAS DE TRICHOMONAS VAGINALIS ISOLADAS DE PACIENTES DA REGIAO DE ARARAQUARA, SP, BRASIL

Trichomonas vaginalis is the flagellate that causes trichomonadiasis, a sexually transmitted disease. Immunological methods have been proposed for the study of antigenic characterization using strains isolated from different patients. This work compares protease profiles from the different strains using gelatin containing polyacrylamide gels to analyse the protease activity. High molecular weight proteases (20 to 100 kDa) were found on gels showing quantitative differences. Human IgG antiproteases were detected by immunoblotting using the same extracts. These proteases could be related with T. vaginalis pathogenesis.