Immuno-proteomic screening of human pancreatic cancer associated membrane antigens for early diagnosis / 中华外科杂志

<p><b>OBJECTIVE</b>To screen and identify the immunogenic membrane antigens in human pancreatic cancer for early diagnosis.</p><p><b>METHODS</b>Membrane protein was extracted from pancreatic cancer cell lines and separated by using 2-DE. One of the two parallel 2-DE gels went for staining while the other underwent immunoblot. Serum IgG, which was purified from clinically collected sera of pancreatic cancer patients, was used as the primary antibodies for the immunoblot. Positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and PMF matching, and then evaluated by bio-informatics methods. The candidate membrane antigens were further validated respectively in cell lines and tissues by RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>The immunoblot of mixed membrane protein with serum IgG from cancer patients showed eight positive dots. These dots were identified with MALDI and PMF as: VDAC-1, VDAC-2, CHCHD3, SLP-2 and TOM40. RT-PCR showed that these membrane antigens were expressed in several pancreatic cancer cell lines. Immunohistochemistry showed prominent SLP-2 over expression in cancer tissue.</p><p><b>CONCLUSIONS</b>VDAC-1, VDAC-2, CHCHD3, SLP-2, and TOM40 are the new candidate immunogenic membrane antigens of pancreatic cancer. These membrane antigens can be subsequently tested in high dangerous population for early diagnosis of pancreatic cancer.</p>

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10Ý a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases

Adverse effect versus quality control of the Fuenzalida-Palacios antirabies vaccine

Avaliamos os componentes da vacina anti-rabica tipo Fuenzalida-Palacios que e ainda utilizada na maioria dos paises em desenvolvimento na imunizacao humana para o tratamento profilatico. Essa vacina e feita em cerebros de camundongos neonatos diluida a 1 por cento, apesar de ser considerada uma vacina com pouco teor de mielina, no entanto ela nao e totalmente livre de mielina, bem como de outros componentes indesejaveis que podem desencadear efeitos adversos a vacinacao, o efeito mais grave esta relacionado com o acidente neuroparalitico pos vacinal associado a sindrome de Guillain Barre. Neste trabalho demonstramos como as vacinas produzidas e distribuidas pelos diversos laboratorios produtores apresentam padroes diferentes de seus componentes com diversos graus de impurezas e com concentracoes proteicas tambem variadas, demonstrando que os processos de producao podem variar em cada laboratorio e que essas diferencas que poderiam ser controladas por meio de um melhor controle de qualidade podem afetar e comprometer a imunizacao acarretando em riscos e efeitos adversos apos a utilizacao dessas vacinas

Characterization and potential uses of rabbit polyclonal antibodies against human plasma lecithin-cholesterol acyltransferase

Familial and secondary deficiency of plasma lecithin-cholesterol acyltransferase (LCAT) produce circulating lipoprotein particles with gross structural and compositional abnormalities; these have adverse effects on a variety of cellular functions. Factors affecting hepatic synthesis and secretion of this plasma enzyme are largely unknown but, potentially, some of them can be investigated with monospecific antibodies. In the present study, enzymically active LCAT was purified 40,000-fold from human plasma and then used to raise polyclonal antibodies in New Zealand White rabbits. Addition of this antiserum (1 mul) to human plasma (25 mul) completely inhibited LCAT activity, although it was less effective against plasma from other species. The antibodies appeared to be monospecific to plasma LCAT. They gave a single precipitin arc by crossed immunoelectrophoresis, while immunodiffusion established that there was no cross-reactivity with several apolipoproteins or with serum albumin. Moreover, the antiserum was successfully used to detect LCAT in normal human plasma by Laurell rocket immunoelectrophoresis. By contrast, Western blotting of plasma proteins using whole LCAT antiserum was largely unsuccessful because of high background staining, although this could be substantially reduced by use of an IgG fraction. However, the whole antiserum readily immunoprecipitated LCAT secreted into the culture medium of HepG2 cells, a human hepatoblastoma cell line, pre-labelled with [35S]methionine, the [(35)S]-labelled LCAT appearing as a narrow 65-kDa protein band by electrophoresis and fluorography. We conclude that polyclonal antibodies may be an important tool to investigate the characteristics and underlying mechanisms of secondary LCAT deficiencies, including those associated with hepatic cirrhosis and schistosomiasis.

Contribuiçäo da imunoeletroforese cruzada em líquido cefalorraquidiano e/ou soro no diagnóstico de infecçöes por Neisseria Meningitidis grupo B no Brasil / Contribution of counterimmunoelectrophoresis in cerebrospinal fluid and/or serum in the diagnosis of Neisseria meningitidis group B infections in Brazil

Imunoeletroforese cruzada (IEC) foi empregada para pesquisar a presença de antígeno de Neisseria Menigitidis grupo B em 185 amostras de líquido cefalorraquidiano (LCR) e/ou 125 amostras de soro. Dentre as amostras de LCR, 103 (55,7 por cento) foram positivas apenas através da cultura e 36 (19,5 por cento) apenas na reaçäo de IEC. Em 34 (18,4 por cento) destas amostras, ambos os métodos apresentaram resultados positivos. Nos casos em que a cultura foi negativa, a IEC em amostras de LCR e/ou soro contribuiu para o diagnóstico da doença meningocócica grupo B em 26,7 por cento dos casos, sendo que em 99,1 por cento deles já havia sido iniciada terapia antibacteriana. Considerando os resultados positivos obtidos apenas na IEC do soro, foi observada uma percentagem de 5,6 por cento. A contribuiçäo da IEC em LCR e/ou soro foi significativa

Avaliaçäo do teste de látex comparativamente à cultura e à imunoeletroforese cruzada no diagnóstico de meningites bacterianas / Evaluation of latex agglutination test in comparison to culture and counterimmunoelectrophoresis in the diagnosis of bacterial meningitis

Um total de 326 amostras de líquido cefalorraquidiano de pacientes com evidência clínica de meningite foi estudado empregando-se um reagente de látex comercial para demonstrar a presença de antígenos bacterianos. As amostras de líquido cefalorraquidiano eram purulentas ou turvas e foram pesquisados antígenos de Neisseria meningitidis, grupos B e C, Haemophilus influenzae b e Streptococcus pneumoniae. Os resultados da detecçäo de antígenos bacterianos através do reagente de látex mostraram um melhor desempenho deste método em comparaçäo à cultura para Neisseria meningitidis grupos B e C e para Streptococcus pneumoniae. Excluindo Streptococcus pneumoniae, para o qual näo foi realizada a imunoeletroforese cruzada, o desempenho do látex mostrou-se superior à imunoeletroforese cruzada apenas para Neisseria meningitidis grupo B. A comparaçäo de resultados do teste de látex com os obtidos através de culturas revelou índice máximo de sensibilidade para Neisseria meningitidis grupo B e para Spreptococcus pneumoniae. Comparando os resultados com os obtidos de imunoeletroforese cruzada, os maiores níveis de sensibilidade foram observados para Neisseria meningitidis grupo B, e para Haemophilus influenzae b. Com relaçäo à especificidade, os valores foram satisfatórios para todos os agentes bacterianos pesquisados, em relaçäo aos outros dois métodos diagnósticos, exceçäo da imunoeletroforese cruzada para Streptococcus pneumoniae que näo foi analisada. O índice de concordância Kappa(K) demonstrou melhores índices K para Haemophilus influenzae b quando comparado com a cultura, e melhores índices K também para este agente bacteriano e para Neisseria meningitidis grupo C, quando os resultados da aglutinaçäo de látex foram comparados com o método de imunoeletroforese cruzada

Analysis of Platelet Membrane Glycoprotein Iib-IIIa Complex in Whole Blood of Glanzmann's Thrombasthenia by Flow Cytometry

Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder characterized by prolonged bleeding time, ad deficient or absent clot retraction in the presence of normal platelet count. The major underlying abnormality in this disease is grossly defective first-phase aggregation of platelet, which are unresponsive to ADP or other platelet agonists such as epinephrine, collagen, thrombin in any concentration. This disability is caused by a decrease or absence of the platelet membrans glycoprotein IIb-IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix fibronectin, and vitronectin On the development of surface labeling technique, a variety of biochemical techniques such as radioimmunoassay, crossed immunoelectrophoresis and SDS-PAGE have been used to study the structure and the function of platelet membrane glycoproteins, and to detect the platelet functional defect. But all of these techniques demand a relatively large amount of homogeneous paletelet population that requires manipulation through isolation and washing procedures before analysis. In order to eliminaste such an intricate procedure, we have applied method for analyzing platelet surface components in whole blood using monoclonal antibody and flow cytometry to recognize the absence of severe reduction of platelet membrane glycoprotien llb-llla complex. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as well as for carriers of this disease. Fow cytometry technique provides a sensitive tool for investigating platelet functional defects caused by altered expression or deficiency of platelet surface proteins.

Antigenic relationship between mugwort and ragweed pollens by crossed immunoelectrophoresis

Mugwort and ragweed pollens have been considered as important respiratory allergens in Korea. These two pollens are abundant in the air of Seoul from August through October. Many ragweed-sensitive patients have shown concurrent sensitivities to mugwort pollen. However the antigenic relationship between these two pollens has not been clarified. To observe the cross-reactivity between them, we developed polyclonal anti-mugwort and anti-ragweed antibodies by immunization on New Zealand white rabbits, and performed crossed immunoelectrophoresis(CIE) with two pollen extracts. Five precipitation lines were formed by mugwort and anti-mugwort antibody. One precipitation line was formed by ragweed and anti-ragweed antibody. There was no reaction from mugwort and anti-ragweed antibody, and from ragweed and anti-mugwort antibody. These results indicate that there is no cross-antigenicity between mugwort and ragweed pollens.

Imunidade humoral em pacientes com fibrose cística / Humoral immunity of patients with cystic fibrosis

Pacientes com FC frequentemente såo acometidos por infecçöes pulmonares, principalmente por P. aeruginosa tanto da variedade nåo mucóide como mucóide. Esta última, quando se instala, é acompanhada geralmente de deterioraçåo progressiva da funçåo pulmonar. Detectam-se nestes pacientes, anticorpos para o microorganismo e diversos produtos por ele produzidos. A imnunoeletroferese cruzada tem sido tradicionalmente utilizada para pesquisa de precipitinas para P. aeruginosa. Esta surgem pouco após o início do episódio infeccioso e guardam correlaçåo com a cronicidade do processo, quando se mostram mais numerosoas. Em pacientes infectados por P. auruginosa mucóide, o número de precipitinas costuma ser maior que nos infectados pela variedade nåo-mucóide. As precipitinas habitualmente nåo surgem nos pacientes apenas colonizados, servindo assim para distinguir casos de colonizaçåo dos de infecçåo, e indicando a oportunidade do emprego de antibioticoterapia nestes pacientes. O número de precipitinas se reduz após controle do processo infeccioso. Outras técnicas vêm sendo desenvolvidas com sucesso, para a pesquisa de anticorpos para P. aureginosa, visando a obtençåo de maior sensibilidade e especificidade, abrindo caminho para novas possibilidades de investigaçåo

Activity of a platelet protein kinase that phosphorylates fibrinogen and histone in Argentine hemorragic fever

La actividad de una proteinoquinasa plaquetaria que fosforila la cadena alfa del fibrinógeno y la histona como substrato exógeno, se evaluo en 28 pacientes con fiebre hemorrágica argentina, agrupados de acuerdo a la forma clínica en: 13 leves, 6 comunes y 9 graves. Las muestras de sangre se obtuvieron antes del tratmiento con plasma inmune, 4 dias después y en la convalescencia. La fosforilación de la histona exógena y del fibrinógeno se estudió con 25 Ci/mmol ( gamma-32p)-ATP. Simultaneamente se efectuó el recuesto de plaquetas y se midió la actividad del interferon alfa (IFN). La fosforilación de la histona se halló por debajo de los valores normales en todos los pacientes durante la fase aguda de la enfermedad. Esta reducción fue coincidente con los titulos más altos de IFN. La fosforilación del fibrinógeno estuvo igualmente disminuida. La fosforilación de la histona y del fibrinógeno estaban aun disminuidos 4 dias después del tratamiento, cuando el IFN era practicamente no dosable. El bajo nivel de fosforilación no puede atribuirse solamente a una disminución del número de plaquetas y podría ser otra evidencia de la existencia de una alteración plaquetaria en los pacientes con fiebre hemorragica argentina

Antigenic analysis of Cysticercus cellulosae by crossed immunoelectrophoresis & its role in immune diagnosis of neurocysticercosis.

The antigenic composition of Cysticercus cellulosae cysts excised from infected pig and autopsied human brain was analysed by crossed immunoelectrophoresis with an intermediate gel technique using rabbit hyperimmune serum. Normal pork muscle and human brain antigen were used to differentiate parasite derived components from that of host. Attempts were made to look for the rich source of parasitic immunodominant antigens by analysing preparations of different parts of cyst namely scolex and fluid using rabbit hyperimmune serum. Twenty three antigenic components were identified in sonicate extract of porcine cyst, of which 15 were parasite derived. On comparison with antigens of whole cyst sonicate, scolex showed 10, cyst fluid 9 and human cyst sonicate 11 parasite derived antigens. Serum and cerebrospinal fluid (CSF) of neurocysticercotic patients reacted with 12 parasite derived antigens of porcine cyst sonicate (PCS) in a heterogenous manner. It was also noticed that human cyst sonicate (HCS) lacked 4 of the parasite derived antigens present in the PCS.

Valor da bacterioscopia, cultura e imunoeletroforese cruzada no diagnóstico das meningites bacterianas / Bacterioscopy, culture and counterimmunoelectrophoresis values in the diagnosis of bacterial meningitis

Em período pós-epidêmico de meningite bacteriana foi planejado comparar a possibilidade do exame bacterioscópico, cultura e pesquisa de antígenos polissacarídeos através da imunoeletroforese cruzada (IEC) no diagnóstico dos três agentes etiológicos que aparecem com maior frequência nestas infecçoes, Neisseria meningitidis, Streptococcus pneumoniae e Haemophilus influenzae. Nas 326 amostras de líquido cefalorraquidiano (LCR) em que foram identificadas estas bactérias, e exame bacterioscópico foi positivo em 273 (83,74%), a IEC em 273 (83,74%) e a cultura em 207 (63,50%). Para o total de amostras estudadas tivemos igual número de casos diagnosticados pela bacterioscopia e IEC (p<0,05); entretanto, quando comparamos a positividade dos métodos laboratoriais associados entre si, a positividade da bacterioscopia para os três agentes citados (95,33%) supera a da IEC (91,05%) e a da cultura (77,04%) (p<0,05). A IEC é um importante método diagnóstico, principalmente quando aplicado em LCR que por qualquer razäo é impróprio à bacterioscopia e cultivo de bactérias. Nenhum dos métodos citados deverá ser utilizado em detrimento do outro; a associaçäo dos métodos além de melhorar as possibilidades diagnósticas evitando erros de interpretaçäo, estabelece controle de quantidade no diagnóstico laboratorial

Alfa-manosidasa: su acción sobre antitrombina III, fibrinógeno y plasminógeno / Alfa-manosidase: its action on antithrombin III, fibrinogen and plasminogen

La alfa-manosiadasa (alfa m) es una hidrolasa ácida que se encuentra en los gránulos azurófilos de los leucocitos polimorfonucleares y en menor concentración en los linfocitos. En leucemias agudas no linfoides sus niveles se hallan muy aumentados respecto de los controles normales (p < 0,001). En estos pacientes, se encuentran alteraciones funcionales e inmunológicas de algunas glicoproteínas del sistema plasmático de coagulación y de fibrinólisis, tales como la antitrombina III (AT III), el fibrinógeno o factor I de coagulación (I) y el plasminógeno (Plg). Debido a esto, investigamos la acción de alfa m sobre estas proteínas purificadas, a partir de plasma humano normal, utilizando métodos inmunoelectroforéticos y electroforesis en gel de poliacrilamida con SDS. Se hallan modificaciones importantes en AT III y I y menores en Plg, similares a las obtenidas en los plasmas de los pacientes. Luego, parece que la acción de hidrolasas ácidas es posible in vivo bajo ciertas condiciones patológicas

Caracterizaçäo imunológica do fator agregador das plaquetas (PAF) no plasma circulante do boi / Immunological characterization of platelet aggregation factor (PAF) in bovine circulating plasma

A metodologia utilizada em experiências anteriores para a purificaçäo e caracterizaçäo do PAF bovino foi também empregada para o estudo da molécula do PAF no plasma circular do boi. Ao contário do que se esperava, a imunoeletroforese cruzada do plasma de boi em gel de agarose a 2% näo forneceu os resultados esperados. A utilizaçäo, poré, de gel de agarose a 1% e a 0,8% permitiu o paparecimento de vários picos de imunoprecipitado com identidade total, tendo o gel a 0,8% fornecido uma resoluçäo maior. A vaiaçäo da distância dos orifícios à linha demarcadora entre o gel superior e o gel inferior foi também estudada. Os melhores resultados foram obtidos quando os orifícios se situavam próximos da linha de separaçäo. Esses achados confirmam os resultados de experiências anteriores e sugerem que o PAF circula no plasma de boi como uma série de agregados de altos pesos moleculares

Precipitating antibodies to non-treated dengue type 2 viral antigens in sera of Thai hemorrhagic fever patients by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of dengue type 2 virus revealed at least two precipitating antigens which shared some antigenic determinants. Glycoprotein components of both antigens were detected by binding to concanavalin A. Sera from dengue hemorrhagic fever patients showed precipitating antibodies to both antigens which could be quantitated according to the precipitate patterns formed in the intermediate gel of crossed immunoelectrophoresis. All secondary dengue hemorrhagic fever patients demonstrated an increase in precipitin titers in convalescence sera. Most patients with mild illness contained precipitating antibodies in acute phase sera whereas severe cases did not. Convalescent sera from severe cases showed only low titers. These precipitating antibodies may be associated with protection since they were produced early only in those with mild form of illness.