Comparison of three vascular endothelial markers in the evaluation of microvessel density in breast cancer

Purpose: To evaluate the microvessel density by comparing the performance of anti-factor VIII-related antigen, anti-CD31 and, anti-CD34 monoclonal antibodies in breast cancer. Methods: Twenty-three postmenopausal women diagnosed with Stage II breast cancer submitted to definitive surgical treatment were evaluated. The monoclonal antibodies used were anti-factor VIII, anti-CD31 and anti-CD34. Microvessels were counted in the areas of highest microvessel density in ten random fields (200 x). The data were analyzed using the Kruskal-Wallis nonparametric test (p < 0.05). Results: Mean microvessel densities with anti-factor VIII, anti-CD31 and anti-CD34 were 4.16 +/- 0.38, 4.09 +/- 0.23 and 6.59 +/- 0.42, respectively. Microvessel density as assessed by anti-CD34 was significantly greater than that detected by anti-CD31 or anti-factor VIII (p < 0.0001). There was no statistically significant difference between anti-CD31 and anti-factor VIII (p = 0.4889). Conclusion: The density of stained microvessels was greater and staining was more intense with anti-CD34 compared to anti-CD31 and anti-factor VII-related antigen.

Anti-Toxocara spp. antibodies in sheep from southeastern Brazil

The purpose of this study was to evaluate the frequency of anti-Toxocara antibodies in sheep from Presidente Prudente, southeastern Brazil. Serum samples were obtained from 365 sheep of diverse breeds and different ages. Samples were collected at a slaughterhouse and at farms located in Presidente Prudente. Three groups of animal of different ages were evaluated according to age: Group I: between 1 and 6 months old; Group II: between 7 and 10 months old; and Group III: between 11 and 15 months old. An ELISA test was carried out to detect anti-Toxocara antibodies (IgG) using the excretory-secretory antigens of Toxocara canis (TES) larvae. In total, 183 out of 365 animals (50.1%) were positive for anti-Toxocara antibodies. The frequency of antibody detection was directly proportional to the age of the animals (p<0.0001). indicating a relationship between infection and aging. In Group III, there was a higher prevalence in females (p = 0.0041). The relevance of these animals to the epidemiology of toxocariasis in pets and human should be considered. (C) 2011 Elsevier B.V. All rights reserved.

Estudo da incidência da infecção por citomegalovírus através da técnica de antigenemia, em uma coorte de pacientes transplantados renais

O citomegalovírus (CMV), está entre os principais agentes infecciosos que acometem pacientes transplantados renais. A infecção por CMV está relacionada ao status sorológico do doador e receptor, bem como o tipo e intensidade da imunossupressão utilizada. A infecção, e em especial a doença citomegálica, determinam aumento da morbi-mortalidade após o transplante. O espectro da doença varia desde formas assintomáticas até a doença sistêmica grave com comprometimento de vários órgãos. A doença por CMV é diagnosticada através da evidência laboratorial de infeção, associada a quadro clínico compatível. A técnica da antigenemia identifica a presença do antígeno viral p65 em leucócitos do sangue periférico através de reação de imunoperoxidase utilizando-se anticorpos monoclonais. O objetivo principal deste trabalho foi o de determinar a incidência de infecção por CMV em uma coorte de pacientes transplantados renais usando a antigenemia como ferramenta diagnóstica. Secundariamente buscou-se avaliar o impacto desta infecção nas sobrevidas dos pacientes e dos enxertos em 6 anos de acompanhamento. No período de inclusão no estudo, janeiro de 1994 a fevereiro de 1995, foram realizados 74 transplantes renais na Santa Casa de Porto Alegre–RS. As amostras de sangue para a detecção da antigenemia foram obtidas semanalmente durante a internação hospitalar e posteriormente, sempre que houvesse suspeita clínica de infecção por citomegalovírus. Das 229 amostras analisadas, 51 (22,3%) foram positivas, em 24 pacientes, dos quais 41,6% (10/24) evoluíram de forma assintomática, 33,3% (8/24) apresentaram sintomas leves, e 25% (6/24) desenvolveram sintomas compatíveis com doença citomegálica. Desta forma, coorte estudada, a incidência de infecção e doença por CMV foram de 33,3% e 8,4%, respectivamente. Não houve associação entre as doses de imunossupressores, o uso de anticorpos monoclonais e número de episódios de rejeição com o desenvolvimento de infecção e doença por CMV. Nos transplantes realizados com doadores vivos, a incidência de infecção por CMV nos receptores de rins de doadores com sorologia positiva foi 61,9%, e nos receptores de doadores com sorologia negativa foi 14,3% (p=0,005). Os transplantes com receptores com sorologia negativa transplantados com rins de doadores soropositivos apresentaram incidência significativamente maior de infecção (75%) e doença (75%) por CMV do que os receptores com sorologia positiva transplantados com órgãos de doadores com soronegativos, 13,3% e 0%, respectivamente (p<0,05). A sensibilidade da antigenemia em detectar os pacientes que desenvolveram doença citomegálica foi de 100% e a especificidade foi 72,7%, com valor preditivo positivo de 25% e valor preditivo negativo de 100%. No grupo de pacientes que apresentou doença por CMV, ao término do seguimento, ocorreu um número significativamente mais elevado de perdas do enxerto (85%) do que no grupo de pacientes em que a infecção foi assintomática (29%), acarretando impacto negativo nas curvas de sobrevida de enxertos e pacientes (LogRank; p<0,05). A antigenemia mostrou ser uma ferramenta diagnóstica importante no manejo dos pacientes transplantados renais, possibilitando o diagnóstico precoce da infecção e auxiliando na identificação dos pacientes infectados que estão sob maior risco de desenvolvimento da doença.

Expressão de p53 e relação clínico-patológica no adenocarcinoma de reto

O adenocarcinoma colorretal é um dos tumores malignos mais freqüentes no mundo ocidental. Sua incidência varia mundialmente; nos Estados Unidos (EUA), é o terceiro câncer mais comum entre os homens e o segundo mais comum entre as mulheres, sendo a segunda causa de morte por câncer, superada apenas pelo tumor de pulmão. No Brasil, está entre as seis neoplasias mais freqüentes, ocupando a quarta posição em mortalidade. Os principais indicadores prognósticos do adenocarcinoma colorretal incluem a diferenciação histológica, profundidade de invasão e ocorrência de metástases. Recentemente, têm sido realizados diversos estudos usando técnicas de biologia molecular objetivando a identificação de novos parâmetros prognósticos. Entre estes, os fatores que regulam o ciclo celular influenciando no crescimento e mecanismo de apoptose têm demonstrado resultados promissores. O p53 é um gene supressor de tumores, localizado no braço curto do cromossomo 17; produz uma proteína chamada p53. Sua principal função é controlar pontos de checagem do ciclo celular, promover o reparo do DNA através do estímulo de outras proteínas (p21, por exemplo) e estimular a apoptose. Mutações deste gene produzem uma proteína p53 inativa que acumula nas células tumorais. A expressão desta proteína alterada é detectada em 30 a 70% dos tumores de reto e pode estar relacionada a mau prognóstico. O p53 é um dos genes mais comumente mutados no câncer humano. O objetivo deste estudo foi correlacionar a expressão imuno-histoquímica da proteína p53 com variáveis clínico-patológicas do adenocarcinoma de reto e sobrevida. Foram estudados 83 casos de pacientes operados no Hospital de Clínicas de Porto Alegre entre 1985 e 1997 através de reação imunohistoquímica utilizando anticorpo monoclonal Pab-1801 em amostras biológicas fixadas em formalina e armazenadas em blocos de parafina. Com um ponto de corte de 5%, 44 pacientes (53%) demonstraram expressão imunohistoquímica da proteína p53 maior que 5% e, com um ponto de corte de 20%, 36 pacientes (43,4%) demonstraram a expressão maior que 20%. Não houve associação estatisticamente significativa entre a expressão de p53 e as variáveis idade, gênero, localização, tamanho do tumor e comprometimento circunferencial. Encontramos associação entre p53 e óbito, recidiva local, metástases e recidiva total quando utilizado o ponto de corte de 20%, indicando um pior prognóstico nos pacientes com p53 positivos. Na análise multivariada em relação à sobrevida, o p53 teve poder prognóstico independente em relação às variáveis da classificação Astler- Coller e grau de diferenciação histológica da neoplasia.

Presence of antibodies against Leishmania chagasi in haemodialysed patients

SOUZA,Roberto Mascarenhas et al.Presence of antibodies against Leishmania chagasi in haemodialysed patients.Transactions of the Royal Society of Tropical Medicine and Hygiene,v. 103, p.749-751, 2009.

Presence of antibodies against Leishmania chagasi in haemodialysed patients

SOUZA, R. M. et al. Presence of antibodies against Leishmania chagasi in haemodialysed patients. Transactions of the Royal Society of Tropical Medicine and Hygiene, v. 103, n.7, p. 749—751. ISSN 0035-9203.

Avaliação dos parâmetros hematológicos e imunofenotípicos de pacientes com doenças linfoproliferativas crônicas no Rio Grande do Norte

Chronic lymphoproliferative disorders (DLPC) are lymphoid system diseases characterized by the abnormal proliferation of mature lymphocytes that affect B cells, T lymphocytes and NK cells. The aim of the study was to demonstrate the relevance of immunophenotyping by flow cytometry in patients with prolonged lymphocytosis and / or cytomorphological changes compatible with lymphoproliferative diseases. In this study 460 patients (244 men and 216 women) with DLPC were evaluated. Were analyzed by flow cytometry with a panel of monoclonal antibodies consisting of CD3, CD4, CD5, CD8, CD10, CD19, CD22, CD23, CD25, CD38, CD45, CD16/CD56, and HLADR heavy and light chains of immunoglobulins. It also examines information regarding age, gender of patients and laboratory data as leucocytes, cytomorphological analysis, platelet count and hemoglobin determination. The results showed 398 cases of chronic lymphoproliferative disorders and 62 of DLPC B cell lymphoproliferative diseases T. B showed the following distribution : 253 cases of chronic lymphocytic leukemia (CLL), 42 cases of multiple myeloma ( MM ), 37 cases of lymphoma non - Hodgkin lymphoma in leukemic phase (NHL) , 17 cases of pro- B lymphocytic leukemia ( B -PLL), 15 cases of mantle cell lymphoma (MCL ), 12 cases of plasma cell leukemia ( PCL), 9 cases of lymphoma Burkitt (Linf B), 8 cases of leukemia villous cells ( LCV), 3 cases of splenic lymphoma with villous cells (LECV), a case of follicular lymphoma (LF) and a Waldenströn macroglobulinemia ( MW). The diseases source NK / T were 23 cases of peripheral T cell lymphoma (LCTP), 14 cases of T prolymphocytic leukemia (T -PLL), 10 cases of leukemia T of large granular lymphocytes (LGL -T) 9 cases of leukemia cells of adult T (LCTA), 5 cases of Sezary syndrome (SS) and a case of large granular NK leukemia (LGL -NK) lymphocytes. In conclusion, the combined use of the monoclonal antibody panel careful cytomorphological analysis was shown to be essential in immune diagnosis and classification of chronic lymphoproliferative disorders. This study was approved by the IRB - HUOL under number 356 / 09

Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica

Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL

Avaliação dos marcadores celulares por citometria de fluxo nos portadores de leucemia mieloide aguda atendidos no Hemocentro do Rio Grande do Norte-Hemonorte

The acute myeloid leukemia (AML) is a disease in which malignant myeloblasts expand, build up and suppress normal hematopoietic activity would represent a major diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these tumors have become more faithful, facilitating the treatment and monitoring of patients. The objectives of this study: diagnosis and classification of AML based on immunophenotyping by flow cytometry with a panel of AcMo specific for acute leukemias, set the frequency of AML in samples from patients with acute leukemias sent to the Department of Hematology Blood Center of Rio Grande do Norte - HEMONORTE, establish standards of antigen expression for different subtypes of acute leukemia and its correlation with the newly diagnosed cases refractory to treatment and recurrence of the disease, standardization of methods for detection and labeling of surface antigens by flow cytometry and intracytoplasmic flow, and observe the frequency of acute leukemia with aberrant phenotypes rare. During the study, 351 were diagnosed acute leukemia, and 179 (51%) classified as AML and 172 (49%) and ALL, which were excluded from the present work. Of the 179 AML, 92 (51.4%) were female and 87 (48.6%) were male, with ages ranging from 3 to 95 years of ag, with higher incidence in individuals in the age group of 41 to 65. Splenomegaly was the clinical finding more present, a total of 147 cases (82.1%), followed by hepatomegaly present in 132 cases (73.7%). The hemorrhagic events were observed in 55 cases (30.7%). Lymphadenopathy in turn was detected in 20 of 179 cases (11.2%). In order to classify subtypes of AML, we used a large panel of monoclonal antibodies, obtaining the following results: AML M0, 02 (1.1%) AML M1, 40 (22.3) AML M2, 60 (33.5) AML M3, 22 (12.3%) AML M4, 10 (5.6) AML M5, 13 (7.3%) AML M6 06 (3.4%) and AML M7 01 (0.6%). We observed some cases with aberrant expression of some antigens such as CD7, CD4, CD19, CD3, CD5 and TdT, CD 7 was present in 30 (16.8%), CD4 in 5 (2.8%), the CD 3 in 5 (2.8%), the CD19 in 3 (1.7%), the CD5 in 3 (1.7%) and TDT was in 7 (3.9%) cases of AML .the CD8 and CD79a was present in only a 1 case.

Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs

O presente trabalho estudou um ensaio imunoenzimático (ELISA) indireto para a detecção de anticorpos anti-Babesia canis no soro de cães, tendo a Reação de Imunofluorescência Indireta (RIFI), como teste de referência O antígeno utilizado no ELISA do presente estudo consistiu em uma preparação antigênica solúvel de merozoítas B. canis e as diluições ótimas do antígeno, soros e conjugado foram determinadas por titulação em bloco, utilizando soros de referência positivos e negativos. A preparação antigênica solúvel de B. canis ótima foi de 10 µg.mL-1, com soros de referência positivos e negativos em uma única diluição de 1:100, e conjugado a 1:4.000. Um total de 246 amostras séricas foram colhidas em cães, durante a campanha de vacinação anti-rábica em Jaboticabal, São Paulo, Brasil e a presença de anticorpos anti-B. canis foi avaliada pelo ELISA e RIFI. Nestas condições, a média de absorbância dos soros de referência negativos foi de 0,129 ± 0,025, resultando em um ponto de corte de 0,323 (Nível de ELISA 3) e a média da absorbância dos soros de referência positivos foi de 2,156 ± 1,187. As amostras com sorologia positiva para B. canis por ELISA e RIFI foram 67,89% (n = 167) e 59,35% (n = 146), respectivamente. Os resultados obtidos sugerem que o ELISA descrito revelou-se um teste sorológico eficaz no diagnóstico da babesiose canina.

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

Serosurvey for antibodies against Brucella abortus and Leptospira interrogans in pampas deer from Brazil

A survey for antibodies against Brucella abortus, and Leptospira interrogans was conducted on 17 pampas deer (Ozotocerus bezoarticus) from Pantanal Matogrossense (State of Mato Grosso do Sul, Brazil) and on 24 pampas deer from Parque Nacional de Emas (State of Goias, Brazil). Antibodies against B. abortus were detected by plate ag glutination, rose Bengal, and complement fixation tests, antibodies against Leptospira interrogans were detected by the microscopic agglutination test. All sera were negative for B. abortus antibodies and all deer sera from Parque Nacional de Emas were negative for L. interrogans antibodies. Four (24%) of 17 sera from Pantanal Matogrossense were positive for L. interrogans serovar (n = 2) hardjo, wolffi (n = 1) and mini (n = 1). While these diseases do not appear to be of major importance to the health status of Pampas deer, it appears that deer are reservoir for leptospirosis in one of the study areas.

Detection of antibodies against bovine respiratory syncytial virus (BRSV) in dairy cattle with different prevalences of bovine herpesvirus type 1 (BoHV-1) in São Paulo State, Brazil

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

Use of near-infrared Raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years for many biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood scrum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.