Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Determination of Carbohydrate-deficient Transferrin Levels by Using Capillary Electrophoresis in a Korean Population / 대한진단검사의학회지

BACKGROUND: Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). METHODS: We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. RESULTS: The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (kappa coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and gamma-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). CONCLUSIONS: The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.

Caracterização da distribuição da isoforma GFAP delta no tecido nervoso: comparação da sua imunoexpressão com a da GFAP e vimentina na glicose e em gliomas

Cinco diferentes isoformas da proteína GFAP já foram identificadas, sendo GFAPa. a isoforma majoritária contra a qual anticorpos de rotina são dirigidos, enquanto que os transcritos da isoforma minoritária GFAPô são vinte vezes menos expressos do que os da GFAPa.. Num estudo recente baseado em material de autópsia, resultados de imunomarcação sugeriram que, no cérebro adulto normal, a GFAPô é especificamente expressa por uma subpopulação astrocitária em áreas subpial e subependimária, e que não estaria envolvida na gliose reativa. Entretanto, estes astrócitos GFAPô positivos são aqueles que normalmente apresentam uma super-expressão de GFAP, sendo marcados também pela vimentina. Resultados negativos não necessariamente indicam ausência dessa isoforma. No presente trabalho, a imunoexpressão da GFAPô foi estudada em espécimes cirúrgicos representativos de diversas formas de gliose e diferentes gliomas do adulto, fixados em condições ótimas com formol-zinco, e comparada com a da GFAP e vimentina. Em todos os processos de gliose testados, assim como no controle de cérebro normal, a imunomarcação de GFAPô foi semelhante à de vimentina, sugerindo que, nestas condições, a imunomarcação de GFAPô depende do nível de GFAP expressa, havendo um limiar abaixo do qual a GFAPô não é detectável. A imunomarcação de GFAPô permitiu observar que a gliose subpial de Chaslin é formada por uma multiplicação dos processos celulares da glia Iimitante, também sugerindo que tanto em condições normais quanto reacionais o estado funcional destes astrócitos e daqueles da camada molecular é altamente variável no espaço e possivelmente no tempo. Nossos dados mostraram que em todos os gliomas testados, a imunorreatividade da GFAPô foi semelhante à da GFAP, porém com um padrão mais variável e menor intensidade de marcação, indicando que este marcador não mostraria maior interesse do que o da GFAP para a subclassificação destes tumores.

Of the 5 isoforms of GFAP proteins already identified, GFAPa represents the major isoform against which current antibodies are directed, whereas GFAPo is a minor isoform, its transcripts being twenty-fold less expressed than that of GFAPa. In a recent study based on autopsy material, immunostaining suggested that, in normal adult human brain, GFAPo is specifically expressed by a subpopulation of astrocytes in subpial and subependymal areas, but is not involved in reactive gliosis. However, these GFAPo positive astrocytes are those which normally overexpress GFAP and stain positively for vimentin. In addition, negative immunostaining does not prove its absence. In the present study, GFAP&-immunostaining was assessed in surgical samples representative of several types of gliosis and different gliomas of the adult, optimally fixed with formalin-zinc, and compared with that for GFAP and vimentin. In all processes of gliosis tested, as in normal brain control, the GFAP&immunostaining paralleled that for vimentin, suggesting that, in both conditions, GFAPo-immunoreactivity depends on the level of GFAP expressed, with a threshold under which GFAPo is not detectable. Nonetheless GFAP&-immunostaining allowed to observe that Chaslin's subpial gliosis is formed by a multiplication of the cell processes of astrocytes of the glia limitans, also suggesting that in both normal and reactive conditions, the functional state of these astrocytes and those of the molecular layer is highly variable in space and likely in time. In all gliomas tested GFAPo-immunostaining was similar to that for GFAP, although showing weaker intensity and a more variable pattern. Our observations suggest that this marker has no potential interest over that of GFAP in the subclassification of these tumors.

Análise do padrão de expressão do produto de PKHD1, o gene mutado na doença renal policística autossômica recessiva / Analysis of the expression pattern of the PKHD1 gene product, mutated in autosomal recessive polycistic kidney disease

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de >440 kDa e aproximadamente 230 kDa, e de aproximadamente 140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. /PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of >440 kDa and 230 kDa, and of about 140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland...

O laboratório no diagnóstico e seguimento da acromegalia / The laboratory in the diagnosis and follow-up of acromegaly

Acromegalia é uma síndrome clínica característica, que na maior parte das vezes resulta de um macroadenoma hipofisário produtor de hormônio de crescimento (GH, growth hormone). A hipersecreçäo tumoral crônica de GH provoca deformidades esqueléticas, distúrbios metabólicos, complicaçöes em vários órgäos e sistemas, e acaba reduzindo a expectativa de vida do paciente. O diagnóstico presuntivo baseia-se nos achados clínicos característicos da doença, com a confirmaçäo vindo através de exames laboratoriais e da avaliaçäo radiológica. Do ponto de vista laboratorial, a abordagem diagnóstica inclui dosagens basais e testes endócrinos que comprovem o excesso de GH, através de dosagens diretas do GH e/ou de fatores circulantes GH-dependentes, cujo melhor exemplo é o fator de crescimento insulina-símile-1 (IGF-1, insulin-like growth factor-7). A adenomectomia transesfenoidal permanece como o tratamento inicial de escolha na acromegalia, mas infelizmente a cura cirúrgica ocorre em menos da metade dos pacientes portadores de macroadenomas. Conseqüentemente, os exames laboratoriais têm um papel muito importante no seguimento dos pacientes após a cirurgia, para definir os critérios de cura e monitorar a atividade da doença durante tratamento complementar com radioterapia ou medicamentos. Neste artigo, revisaremos os exames laboratoriais mais freqüentemente utilizados no diagnóstico da acromegalia e alguns métodos experimentais que vêm sendo testados na sua abordagem. Na parte final, apresentaremos as principais recomendaçöes de dois workshops internacionais realizados nos últimos anos com o objetivo de padronizar a avaliaçäo diagnóstica e a conduta terapêutica na acromegalia.

Immunoreactivity of CD99 in Stomach Cancer

CD99 is characteristically expressed in Ewing's sarcoma/primitive neuroectodermal tumor. Recently its immunoreactivity has also been reported in other tumors. However, the significance of CD99 isoforms expressed in these tumors has not been elucidated. In this study, we evaluated the expression of CD99 isoforms and its relationship with histopathologic parameters in gastric adenocarcinomas. Paraffin sections of 46 gastric adenocarcinomas were stained with an anti-CD99 monoclonal antibody, YG32. Twelve (26.1%) cases of 46 gastric adenocarcinomas showed immunoreactivity to YG32. The CD99 expression was also seen both in non-neoplastic foveolar epithelial cells and infiltrating lymphocytes. In addition, Western blot and RT-PCR analyses revealed that the type I is the predominant isoform of CD99 in non-neoplastic and neoplastic gastric tissues. The CD99 expression was usually seen in the intestinal type adenocarcinoma, while rarely in the diffuse type. The CD99 immunoreactivity decreased in MMP-2-overexpressing adenocarcinomas (p=0.028). Our results suggest that the type I is the major isoform of CD99 expressed in non-neoplastic gastric mucosa and gastric adenocarcinomas and its downregulation in gastric adenocarcinoma may be associated with cellular dedifferentiation and/or MMP-2 overexpression.

Hamartomatous gastric polyposis in a patient with tuberous sclerosis

A 42-year-old female diagnosed with tuberous sclerosis was found to have multiple polyps in the fundus of stomach. On histologic examination, the lesions were hamartomatous polyps. In tuberous sclerosis, many lesions occur in multiple organs and there are several reports about the frequent association of hamartomatous polyps of the colon. However, gastric manifestation of tuberous sclerosis has not been established probably due to its asymptomatic nature. This is the first report of multiple gastric hamartomatous polyposis in patient with tuberous sclerosis.

Hamartomatous gastric polyposis in a patient with tuberous sclerosis

A 42-year-old female diagnosed with tuberous sclerosis was found to have multiple polyps in the fundus of stomach. On histologic examination, the lesions were hamartomatous polyps. In tuberous sclerosis, many lesions occur in multiple organs and there are several reports about the frequent association of hamartomatous polyps of the colon. However, gastric manifestation of tuberous sclerosis has not been established probably due to its asymptomatic nature. This is the first report of multiple gastric hamartomatous polyposis in patient with tuberous sclerosis.