Expressão citofotométrica dos marcadores tumorais CD-34 e fator VIII no câncer de cólon / Cytophotometric expression of the tumor markers CD-34 and factor VIII in colorectal cancer

OBJETIVOS: Verificar a posssibilidade de quantificar a expressão dos marcadores tumorais CD-34 e Fator VIII no câncer de cólon; verificar se existe superioridade entre um marcador e outro para estudo da angiogênese; verificar se há correlação na análise do índice de marcagem e a densidade óptica média nos marcadores utilizados. MÉTODOS: Dezessete casos de adenocarcinoma colorretal recuperados de blocos de parafina e confirmados pela hematoxilina-eosina, foram submetidos à coloração imunoistoquímica pelo método da estreptoavidina-biotina-peroxidase e utilizados os marcadores tumorais CD-34 e Fator VIII. Após este processo as lâminas foram submetidas à leitura no sistema Samba 4000® e avaliadas pelo software Immuno®. Os parâmetros estudados foram: índice de marcagem e densidade óptica, expressos por médias, medianas, valores mínimos, valores máximos e desvios-padrão, analisados estatisticamente. RESULTADOS: Para o marcador CD-34 não houve normalidade dos dados em relação ao índice de marcagem e houve para a densidade óptica. Para o Fator VIII, houve normalidade de dados em relação ao índice de marcagem e para a densidade óptica. CONCLUSÃO: Foi possível quantificar a expressão dos marcadores tumorais CD-34 e Fator VIII através do índice de marcagem e da densidade óptica média; não houve diferença entre os marcadores em relação à média do índice de marcagem e da densidade óptica, não sendo possível definir superioridade entre um e outro; não foi observada tendência à correlação quando comparados densidade óptica e índice de marcagem do Fator VIII e do CD-34 isoladamente estudados; não houve correlação entre o índice de marcagem do Fator VIII quando comparado com o CD-34, bem como a densidade óptica do Fator VIII com o CD-34.

OBJECTIVES: In colorectal cancer, to describe the cytophotometric expression of the CD-34 and Factor VIII; to evaluate the degree of correlation between them; and to compare the CD-34 and Factor VIII expressions in relationship to label index and optical density. METHODS: Seventeen cases of colorectal adenocarcinoma recovered from paraffin-embedded archival tissue and confirmed by hematoxilin-eosin staining, were submitted to streptavidin-biotin-peroxidase immunohistochemical method. In this process was used the tumor markers CD-34 and Factor VIII. The obtained slides were analysed using the SAMBA 4000® system with Immuno® software. The results were evaluated into two parameters: label index and optical density, and expressed by averages, medians, minimum values, maximum values, and standard deviation values. The normality condition of the quantitative variables was investigated by using the Shapiro-Wilks test. In order to evaluate the degree of association between the expressions of the markers, Pearson's Correlation Coefficient or Spearman's Correlation Coefficient were applied. To evaluate the comparison degree of the markers expression, Student's t test or Wilcoxon's no parametric test were used. RESULTS: For the CD-34 there was no data normality for the label index and there was normality in the optical density. For the Factor VIII, there was data normality for the label index and for the optical density. CONCLUSION: When the expressions of CD-34 and Factor VIII markers were correlated, there was no difference between them in relationship to the average label index and average optical density. When the expressions of the CD-34 and Factor VIII were compared, there was no correlation between the two variables.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via alphavbeta3 and alpha5beta1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.

Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.

Determinación de hepatitis C en productos hemoderivados / Hepatitis C determination in blood derivates products

El control de hemoderivados en Venezuela se inició hace más de veinte años y ha sido actualizado a medida que han progresado las investigaciones en el área. Por tanto, ante el riesgo de transmisión de Hepatitis B, C y VIH, se comenzó a realizar el despistaje de estos agentes en hemoderivados empleando las técnicas de detección en muestras de sangre, por lo que el objetivo de este trabajo es demostrar su aplicabilidad en el control del producto final. Los productos analizados fueron albúminas, gammaglobulinas, factor VIII, globulinas específicas tanto puras como realizando diluciones, a fin de evaluar posibles interacciones no específicas. Los resultados demuestran la aplicabilidad de estos kits a nivel del producto final y su importancia, como herramienta para garantizar la calidad de los hemoderivados importados y producidos en el país