Development of a flow cytometry method for detection of bovine multi-cytokines / 生物工程学报

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Efecto de la inhibición de la proteína símil angiopoyetina tipo 3 (ANGPTL3): utilidad para el tratamiento de las dislipemias graves / Angiopoietin-like type 3 (angptl) inhibition: its role in the treatment of severe dyslipidemia

Las variantes de ANGPTL3 con pérdida de función están asociadas con efectos beneficiosos sobre el metabolismo lipídico y de carbohidratos y con riesgo reducido de enfermedad coronaria. Los cambios beneficiosos en los parámetros lipídicos que se obtienen con la inhibición de ANGPTL3 junto con la reducción en aterosclerosis que se observa en modelos animales y en estudios epidemiológicos de genética humana hacen de ANGPTL3 un nuevo objetivo terapéutico para prevenir las enfermedades cardiovasculares. Dos estrategias novedosas han surgido para inhibir esta proteína: un anticuerpo monoclonal y un oligonucleótido antisentido, con capacidad para reducir tanto el colesterol como los triglicéridos plasmáticos en forma notoria. Aunque el horizonte es promisorio, todavía no sabemos si los efectos de una variante presente desde el comienzo de la vida serán reproducidos por la inhibición de esta proteína que se realiza más tarde en la vida a través de una intervención farmacológica. (AU)

Loss-of-function ANGPTL3 variants are associated with beneficial effects on carbohydrate and lipid metabolism, and reduced risk of coronary heart disease. The beneficial changes in lipid parameters obtained by ANGPTL3 inhibition together with atheroprotection observed in animal models and in epi-demiological studies of human genetics make ANGPTL3 a new therapeutic target to prevent cardiovascular diseases. Two novel strategies have emerged to inhibit this protein: a monoclonal antibody and an antisense oligonucleotide, with the ability to significantly lower plasma cholesterol and triglycerides. Although the horizon is promising, we still do not know if the effects of a variant present from the beginning of life will be reproduced by the inhibition of this protein that takes place later in life through a pharmacological intervention. (AU)

Anti-placental growth factor antibody ameliorates hyperoxia-mediated impairment of lung development in neonatal rats

This study investigates the effect of the overexpression of the placental growth factor (PGF) and hyperoxia on lung development and determines whether anti-PGF antibody ameliorates hyperoxia-mediated impairment of lung development in newborn rats. After exposure to normoxic conditions for seven days, newborn rats subjected to normoxia were intraperitoneally or intratracheally injected with physiological saline, adenovirus-negative control (Ad-NC), or adenovirus-PGF (Ad-PGF) to create the Normoxia, Normoxia+Ad-NC, and Normoxia+Ad-PGF groups, respectively. Newborn rats subjected to hyperoxia were intraperitoneally injected with physiological saline or anti-PGF antibodies to create the Hyperoxia and Hyperoxia+anti-PGF groups, respectively. Our results revealed significant augmentation in the levels of PGF and its receptor Flt-1 in the lung tissues of newborn rats belonging to the Normoxia+Ad-PGF or Hyperoxia groups. PGF overexpression in these groups caused lung injury in newborn rats, while anti-PGF antibody treatment significantly cured the hyperoxia-induced lung injury. Moreover, PGF overexpression significantly increased TNF-α and Il-6 levels in the bronchoalveolar lavage (BAL) fluid of the Normoxia+Ad-PGF and Hyperoxia groups. However, their levels were significantly reduced in the BAL fluid of the Hyperoxia+anti-PGF group. Immunohistochemical analysis revealed that PGF overexpression and hyperoxia treatment significantly increased the expression of the angiogenesis marker, CD34. However, its expression was significantly decreased upon administration of anti-PGF antibodies (compared to the control group under hyperoxia). In conclusion, PGF overexpression impairs lung development in newborn rats while its inhibition using an anti-PGF antibody ameliorates the same. These results provided new insights for the clinical management of bronchopulmonary dysplasia in premature infants.

Immunohistochemical analysis of retinoblastoma cell phenotype using neuronal and glial cell markers / Análise imuno-histoquímica do fenótipo de células de retinoblastoma utilizando marcadores de células neuronais e gliais

ABSTRACT Purpose: The cellular origin of retinoblastoma is uncertain as constituent tumor cells heterogeneously express markers of both immature and mature retinal cells. An immunohistochemical analysis of cellular origin may yield valuable insights into disease progression and treatment options. This study aimed to determine the cellular origin of retinoblastoma in a large case series and correlate these findings with histopathological prognostic factors. Methods: Thirty-nine retinoblastoma cases were histopathologically diagnosed and analyzed by immunohistochemistry using monoclonal antibodies against the immature neural cell marker SRY-box containing gene 2 (SOX-2), the mature neuronal cell marker microtubule-associated protein 2 (MAP2), and the mature glial cell marker glial fibrillary acidic protein (GFAP). Histopathological features were also evaluated, including patterns of growth, differentiation, vitreous seeding, and choroidal/scleral, optic nerve, and anterior chamber invasion. Two retinoblastoma cell lines, WERI-1 and Y79, were studied by immunocytochemistry using the same antibodies. Results: Expression of SOX-2 was strong in 97.4% of retinoblastoma cases, while MAP-2 was expressed in 59% of cases. Immunostaining for GFAP was positive only in reactive stromal astrocytes interspersed amongst tumor cells and in peritumoral tissue. There was no correlation between histopathological prognostic factors and immunohistochemical markers. Retinoblastoma cell lines showed strong positivity for SOX2 (90% of WERI-1 cells and 70% of Y79 cells) and MAP2 (90% of cells in both lines). GFAP was completely negative in both cell lines. Conclusion: The majority of retinoblastomas and both RB cell lines expressed an immature neural and/or a mature neuronal cell marker, but not a glial marker. These results indicate a typical neuroblast or neuronal origin and eliminate astrocyte differentiation from neural stem cells as the source of retinoblastoma.

RESUMO Objetivos: Este estudo visa determinar a origem do retinoblastoma em um número de casos e correlacionar essos achados com fatores prognósticos e histopatológicos conhecidos. Métodos: Trinta e nove casos de retinoblastoma foram diagnosticados e analisados com imuno-histoquímica usando marcadores de anticorpos monoclonais contra as células de retina imaturas (SOX-2: SRY-box containing gene 2), contra as células da retina maturas (MAP2: microtubule -associated protein 2) e contra as células gliais maturas (GFAP: glial fibrillar acidic protein). Foram avaliadas características microscópicas dos casos (grau de diferenciação, presença de semeadura vítrea, invasão de coroide/esclera, nervo óptico e câmara anterior). Duas linhas celulares de retinoblastoma (WERI-1 e Y79) também foram testadas, utilizando os três marcadores. Resultados: A expressão de SOX-2 foi positiva em 97,4% dos casos de retinoblastoma, enquanto MAP2 foi positivo em 59% dos casos. GFAP foi apenas positivo no estroma (astrócitos reativos). Não houve correlação entre preditores histopatológicos e marcadores imunohistoquímicos avaliados. As linhagens celulares mostraram positividade para SOX-2 (90% em WERI-1 e 70% das células Y79). Ambas as linhagens celulares se mostraram fortemente positivas con MAP2 (90%), enquanto não houve expressão de GFAP em nenhuma das linhas celulares estudadas. Conclusões: A maioria das células de retinoblastoma desta série de casos expressa marcadores de células retinianas imaturas, além de marcadores de células maduras. As linhas celulares Y79 e WERI-1 apresentaram imunomarcação para ambos os marcadores neurais em percentagens semelhantes a dos casos avaliados. Portanto, estes resultados confirmam a origem neural do tumor em particular. Alem disso, a ausência de células positivas para GFAP no tumor descarta diferenciação de astrócitos em retinoblastoma.

La Escuela de Salud Pública de México: innovación educativa y tecnológica en el nuevo milenio / School of Public Health of Mexico: Educational and technological innovation in the new millennium

Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.

This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.

Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients

Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain β1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the β2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.

Cytokeratin expression in corneal dystrophies / Expressão de citoqueratinas em distrofias corneanas

PURPOSE: To identify an immunohistochemical pattern of epithelial markers in granular, lattice and Avellino corneal dystrophies. METHODS: Twenty-two corneal buttons, diagnosed as lattice (17), Avellino (4) and granular (1) underwent immunohistochemical studies of cytokeratins (CKs) on pa- raffin-embedded sections (group I). Monoclonal antibodies for pan-CK (AE1/AE3) and CKs 3/12, 5/6, 8, 18 and 19 were used. Twenty-two normal corneas were used as the control (group II). RESULTS: Six lattice and 2 Avellino cases of group I stained positively with anti-CK 3/12 in corneal epithelium and areas of corneal stroma deposits. One of these cases of lattice was positive for anti-pan-CK (AE1/AE3) also in the epithelium and areas of corneal stroma deposits with a similar pattern. None of the controls (group II) revealed any staining in corneal stroma. All disease and control cases (groups I and II) revealed positive staining in corneal epithelium. CONCLUSION: AE1/AE3 and CK 3/12 anti-CK positive markers in the stromal deposits of lattice and Avellino dystrophies may suggest an epithelial genesis of the disease.

OBJETIVO: Investigar a expressão de citoqueratinas (CKs) em córneas com distrofias corneanas tipo granular, lattice e Avellino. MÉTODOS: Vinte e dois botões corneanos com diagnóstico anatomopatológico de distrofia estromal tipo lattice (17), Avellino (4) e granular (1) foram submetidos à avaliação imunohistoquímica nos tecidos inclusos em parafina (grupo I). Anticorpos monoclonais para pan-CK (AE1/AE3) e CKs de números 3/12, 5/6, 8, 18 e 19 foram utilizados. Vinte e dois botões corneanos normais foram usados como controle (grupo II). RESULTADOS: Oito casos do grupo I (seis lattice e dois Avellino) apresentaram reações imuno-histoquímicas positivas com anti-CK 3/12, tanto no epitélio como nos depósitos estromais e um destes casos (lattice) também se mostrou positivo para anti-pan-CK (AE1/AE3) com o mesmo padrão de reação. Nenhum caso do grupo II mostrou reação imuno-histoquímica positiva no estroma corneano. Na avaliação imuno-histoquímica dos grupos I e II, o epitélio apresentou uma reação positiva com o anticorpo anti-pan-CK (AE1/AE3) e com o anti-CK 3/12. CONCLUSÃO: O fato da pan-CK e CK 3/12 apresentarem uma reação positiva nos depósitos das distrofias tipo lattice e Avellino sugere uma origem epitelial desses depósitos corneanos.

Trastuzumabe: mecanismos de resistência e possibilidades terapêuticas em câncer de mama HER-2 positivo

O receptor do fqtor de crescimento epidérmico humano tipo 2 (HER-2) é um receptor trasnsmembranacom atividade tirosina quinase, tal molécula encontra-se hiper-expressa em aproximadamente 20% a 25% dos carcinomas invasivos de mama. Os pacientes que apresentam esta característica molecular apresentam padrão de sensibilidade a agentes antineoplásicos diferente dos outros tumores e também um pior prognóstico. O trastuzumabe (Herceptin, Genentech) é um anticorpo monoclonal humanizado do tipo IgG1, direcionado à porção extracelular do HER-2. A adição do trastuzumabe à quimioterapia resulta aumento na sobrevida das pacientes, seja na doença metastática ou no tratamento adjuvante. Infelizmente, uma importante fração destas pacientes não atinge resposta à terapia inicial com trastuzumabe e a vasta maioria daquelas que responde inicialmente desenvolverão resistência em um perído de um ano. Nesta revisão, iremos discutir os mecanismos moleculares que levam à resistência a este agente, assim como as possibilidades terapêuticas que emergem deste conhecimento.

The human epidermal growth factor receptor 2 (HER-2) is a transmembrane receptor with tyrosine-kinase activity overexpressed in about 20-25% of invasive carcinomas of the breast. Patientes with such tumors have different responses to therapeutic agents and a worse outlook, with reduced progression-free and overall survival, when compared with patients harboring HER-2 negative tumors. Trastuzumab (Herceptin, Genentech) is a humanized IgG1 monoclonal antibody against the extracellular domain of HER-2. The combined use of trastuzumab and chemotherapy has resulted in an increase in overall survival rates in the metastatic setting. Unfortunately, a sizable fraction of patients do not respond to initial therapy with trastuzumab, either as a single agent or in combination with chemotherapy, and the vast majority of patients initially responding eventually develop resistance to treatment within 1 year. This review will discuss several molecular mechanisms that can lead to development of trastuzumab resistance, as well as the possibility of exploring these aberrations as therapeutic targets that could help avoid or overcome resistance to trastuzumab, thus enhancing the therapeutic arsenal and the life expectancy of patients with HER2-positive breast cancer.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.

Bovine respiratory syncytial virus: immunohistochemichal detection in mouse and bovine tissues using a Mab against human respiratory syncytial virus

An immunoistochemical (IHC) test was developed to detect bovine respiratory syncytial virus (BRSV) in cell cultures and tissues of experimentally infected mice and calves, using a commercial monoclonal antibody (Mab) against human respiratory syncytial virus (HRSV), as a less expensive alternative, instead of producing specific monoclonal antibodies to BRSV. Clinical samples from calves suffering respiratory disease were also submitted to this test. IHC detected BRSV antigens in mouse tracheas (3, 5 and 7 days post-infection) and lungs (5 and 7 days post-infection), and in one of three lungs from experimentally infected calves. Lungs samples from two naturally infected calves were tested and resulted positive for BRSV by the IHC test. These results suggest that this test may be used in the future for diagnosis as well as a useful tool to assess the distribution of BRSV infections in Brazilian herds.

Desenvolveu-se um teste de imunohistoquímica (IHQ) para detecção do vírus respiratório sincicial bovino (BRSV) multiplicado em cultivo celular e em tecidos de camundongos e bezerros infectados experimentalmente, utilizando um anticorpo monoclonal comercial contra o vírus respiratório sincicial humano (HRSV), como uma alternativa para eliminar os custos de produção de anticorpos monoclonais específicos para o BRSV. Amostras clínicas de bezerros com sintomatologia respiratória foram analisadas. A técnica mostrou-se eficiente na detecção de antígenos do BRSV em traquéias (3, 5 e 7 dias pós-infecção) e pulmões (5 e 7 dias pós-infecção) dos camundongos infectados e em uma das três amostras de pulmões dos bezerros infectados experimentalmente. Amostras de pulmões de dois animais com infecção natural foram positivas para BRSV. Conclui-se que o teste de IHQ pode ser usado no diagnóstico das infecções por BRSV e na avaliação da distribuição dessas infecções nos rebanhos bovinos brasileiros.

Influence of essential fatty acid deprivation on thrombocyte aggregation in rainbow trout

Many reports have indicated that differences in blood cells formation and function in fish could be of dietary origino Essential fatty acids are certainly connected with these cells by virtue of being a source of important compounds like eicosanoids, platelet activating factor in mammals, as well as the cell membrane phospholipids. The thrombocytes from fish fed the commercial diet containing adequate amount of essential fatty acids exhibited greater capacity for aggregation when induced by Collagen Type I(56%), however, this capacity was reduced when fish were fed the essential fatty acids deficient diet (37%). The results obtained in this study demonstrated that EFA exert influence in thrombocytes by affecting their aggregation capacity.

Indicações sobre alterações na formação das células sanguíneas e em suas funções nos peixes têm sido relatadas em diversos trabalhos. Os ácidos graxos essenciais (EFA) certamente estão ligados a essas células devido ao fato de constituírem fonte de componentes importantes, como os eicosanoides, fatores de ativação de plaquetas nos mamíferos, bem como de fosfolipídios de membrana. Trombócitos oriundos de peixes alimentados com uma dieta comercial contendo quantidades adequadas de EFA mostraram uma grande capacidade de agregação quando induzidos por colágeno do tipo I (56%), contudo, essa capacidade encontrou-se reduzida quando os peixes foram alimentados com uma dieta deficiente em EFA (37%). Os resultados obtidos nesse estudo demonstraram que os ácidos graxos essenciais exercem influência nos trombócitos afetando sua capacidade de agregação.

CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells

CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.

Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors

We concentrated ourselves to evaluate the prognostic significance of the p53 gene mutations, its protein expression and MIB-1 index as a proliferative marker in canine mammary tumors. In the present study, a total of 20 cases were examined, among which there were 5 malignant mixed tumors, 4 mammary gland adenocarcinomas, 1 papillary adenocarcinoma, 8 benign mixed tumors and 2 mammary gland adenomas. Positive immunostaining for p53 with PAb240 antibody was found in 2 benign (20%) and 3 malignant (30%) tumors. However, PAb421 antibody did not give positive result at all. In Western blot analysis, the p53 expression in benign and malignant tumors was detected in 4 and 3 cases, respectively. p53 mutations were found in 6 cases out of the cases with detected p53 protein expression. The MIB-1 index in benign and malignant tumors were 17.6+/-20.8% and 29.0+/-27.2%, respectively and there was no significant difference between tumor types. There was a significant correlation between p53 mutations and p53 overexpression (correlation coefficient = 0.5, p < 0.05). In Kaplan-Meier survival analysis, the p53 index was associated with significantly shortened survival time (p < 0.01). In multivariate analysis, p53 overexpression was only an independent factor for indicator of worse prognosis in canine mammary tumors (p = 0.01). These results demonstrated that p53 gene mutations and protein overexpression using the PAb240 anti-p53 antibody were useful predictors of increased malignant potential and poor prognosis in canine mammary tumors.

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.

Distribution of pectins in the pollen apertures of Oenothera hookeri: velans ster/+ster

Cell wall pectins are some of the most complex biopolymers known, and yet their functions remain largely mysterious. The aim of this paper was to deepen the study of the spatial pattern of pectin distribution in the aperture of Oenothera hookeri.velans ster/+ster fertile pollen. We used "in situ" immunocytochemical techniques at electron microscopy, involving monoclonal antibodies JIM5 and JIM7 directed against pectin epitopes in fertile pollen grains of Oenothera hookeri.velans ster/+ster. The same region was also analyzed by classical cytochemistry for polysaccharide detection. Immunogold labelling at the JIM7 epitope showed only in mature pollen labelling mainly located at the intine endo-aperture region. Cytoplasmic structures near the plasma membrane of the vegetative cell showed no labelling gold grains. In the same pollen stge the labelling at the JIM5 epitope was mostly confined to a layer located in the limit between the endexine and the ektexine at the level of the border of the oncus. Some tubuli at the base of the ektexine showed also an accumulation of gold particles. No JIM5 label was demonstrated in the aperture chamber and either in any cytoplasmic structure of the pollen grains. The immunocytochemical technique, when compared with the traditional methods for non-cellulose polysaccharide cytochemistry is fare more sensitive and allows the univocal determination of temporal and spatial location of pectins recognized by the JIM7 and JIM5 MAbs.

Hepatocyte Expressions in Hepatocellular Carcinomas, Gastrointestinal Neoplasms, and Non-neoplastic Gastrointestinal Mucosa: its Role as a Diagnostic Marker

We performed immunohistochemical staining against Hepatocyte (Hep) and CD10 antibodies in 75 hepatocellular carcinoma (HCC), 50 cholangiocarcinomas, 49 colorectal adenocarcinomas, and 308 gastric adenocarcinomas by tissue array method. We also evaluated the various non-neoplastic adult tissues and fetal digestive organs. Hep was expressed in 80% of HCCs, and HCCs without Hep expression were more likely to have a higher Edmondson & Steiner grade than HCCs with Hep expression (p=0.004). In non-HCCs, 16% of cholangiocarcinomas, 8.2% of colorectal carcinomas, and 44.2% of gastric carcinomas expressed Hep. Gastric carcinomas with Hep expression were significantly associated with early gastric carcinomas (p<0.001). In non-neoplastic tissues, Hep was found expressed in normal hepatocytes, small intestinal mucosa, and intestinal metaplasia of the stomach. Fetal hepatocytes expressed Hep after 19 weeks of gestation. CD10 was detected in 46.7% (35/75) of HCCs, and canalicular staining pattern was predominant in HCCs. In conclusion, the expression of Hep and CD10 may help to distinguish HCCs from non-HCCs.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99 percent with rBoIFN-alphaC and by 84 percent with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed

Produçäo de anticorpos monoclonais contra dehidroepiandrosterona (DHEA) e seuemprego no desenvolvimento de ensaios para a dosagem sérica de DHEA-sulfato (DHEA-S) / Monoclonal antiboidies against dehtdroepiandrosterone(DHEA) and their use in the development of assays for serum measurement of DHEA-sulphate (DHEA-S)

Neste trabalho, descrevemos nossa experiência na produçäo e caracterizaçäo de anticorpos monoclonais contra o sulfato de dehidroepiandrosterona (DHEA-S), sua caracterizaçäo e seu uso no desenvolvimento de radioimunoensaios tradicionais, mediante a comparaçäo dos resultados obtidos com um radioimunoensaio clássico empregando anticorpo policlonal. Foram caracterizados três anticorpos monoclonais (H6P4, B2P2 e G10p6) que apresentam coeficientes de afinidade entre 4 x 108 e 1,6 x 109 L/M e especificidades variáveis. A comparaçäo entre os valores obtidos com os três anticorpos monoclonais e aqueles obtidos com o ensaio tradicional mostrou excelente grau de correlaçäo. Os valores obtidos com o anticorpo monoclonal B2P2 apresentaram melhor índice de correlaçäo. Estudos adicionais mostraram que as características do ensaio, que empregou o anticorpo monoclonal B2P2, säo semelhantes àquelas do ensaio tradicional com anticorpo policlonal. Os anticorpos monoclonais obtidos podem servir de base para ensaios mais simples para a dosagem sérica de DHEA-S

Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g) production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC) mAb (W6/32) suppressed lymphocyte proliferation by 90 percent in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67 percent and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60 percent, while in the other 2 cultures IFN-g levels were 36 and 10 percent lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs) or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens