Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW+ vessels. The most common cells were CD68+ macrophages, CD20+ B-cells and CD4+ T-cells. A significant linear correlation between CD4+ and CD20+ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106 percent increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95 percentCI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Inmunogenicidad de los concentrados de Factor VIII doblemente inactivados producidos en UNC-Hemoderivados / Immunogenecity of double inactivation FVIII concentrates manufactures by UNC-Hemoderivados

El tratamiento de elección en la hemofilia A es la administración de concentrados de factor VIII y hasta un 33 % de estos pacientes pueden desarrollar inhibidores dirigidos contra el factor infundido. Varias causas pueden estar involucradas en este proceso inmune siendo la intensidad de la terapia y tipo de concentrados empleados uno de los más estudiados. Por lo tanto, el análisis cuali / cuantitativo de diferentes proteínas que hacen a un concentrado más inmunogénico que otros cobra vital importancia. En este trabajo se evaluaron los niveles de factor VIII antígeno (FVIII:Ag), factor von Willebrand (FvW) funcional e inmunológico, factor de crecimiento transformador ß1 (TGF-ß1), fibrinógeno e inmunoglobulinas G, A y M en diferentes lotes de concentrados de factor VIII manufacturados en UNC-Hemoderivados y fueron comparados con otros productos comerciales de similares características. Se estudiaron 6 lotes de 250 UI y 2 de 500 UI producidos en UNC-Hemoderivados, dos lotes de 250 UI producidos por las firmas A y B y un lote de 500 UI comercializado por la firma C. Para las determinaciones de factor VIII/factor de von Willebrand, funcional y antigénico, se emplearon métodos estándares y para el TGF-ß1 se utilizó un ELlSA comercial. Los resultados obtenidos mostraron que las relaciones entre FVIII:C/FVIII:Ag y FVIII:C/FvW funcional fueron en promedio 0.60 y 0.52 para el producto de UNC-Hemoderivados y 0.50 y 0.38 para los productos de origen comercial. Los valores TGF­ß1 y la actividad específica arrojaron mejores resultados en el producto de UNC-Hemoderivados que los productos A, B y C y en las otras proteínas analizadas no hubo diferencias. Por lo tanto podemos concluir que los concentrados de FVIII producidos en UNC-­Hemoderivados presentan propiedades que potencialmente indicarían una menor respuesta inmune contra el FVIII infundido.

Replacement therapy using plasma factor VIII (FVIII) concentrates is currently the major mean of preventing and controlling bleeding in hemophilia A patients. However. approximately a 33% of these patients may develop inhibitors to the substituted FVIII. Several causes may be involved in the immune process being the intensity of therapy and type of concentrate used one of the most studied. Therefore. the study quali/quantitative analysis of different proteins which make a concentrated more immunogenic than other takes a vital importance. In this study we evaluated FVIII antigen (FVIII: Ag), von Willebrand factor (vWF) functional and immunological, TGF-Beta1, fibrinogen and immunoglobulins G, A and M levels in FVIII concentrates manufactured in UNC-Hemoderivados and compared with other commercial products of similar characteristics. We studied 6 lots of 250 IU and 2 lots of 500 IU manufactured in UNC-Hemoderivados, 2 lots of 250 IU produced by two pharmaceutical companies named A and B and 1 batch of 500 IU marketed by C. For the determinations of F VIII/FvW functional and antigenic we used standards methods and the TGF-ß1 was assayed by ELlSA test. The results show that the relationship between FVIII/FVIII:Ag and FVIII/vWF functional were in average 0.6 and 0.52 for the product of UNC-Hemoderivados and 0.5 and 0.38 for products from commercial sources. TGF-ß1 levels and the specific activity showed upper values in UNC-Hemoderivados compared to commercial products and none difference was observed in the other proteins assayed. Therefore we can conclude that FVIII concentrates produced at UNC-Hemoderivados have properties that indicate a potentially lower immune response against the infused FVIII.

Dosagem plasmática do fator de Von Willebrand e variáveis clínicas como fatores prognósticos no câncer de mama / Plasmatic Von Willebrand factor measurement and clinical variables as prognostic factors of breast cancer

Introdução: O fator Von Willebrand (vW) é uma glicoproteína cujo aumento da concentração sérica está associado ao mecanismo de estimulação do epitélio endotelial facilitando a metástase do câncer de mama.Objetivo: O objetivo deste estudo é avaliar o valor prognóstico do vW no câncer de mama, assim como variáveis clínicas.Métodos: Analisou-se uma coorte de 50 pacientes com câncer de mama (CM) do Serviço de Mastologia do Hospital de Clínicas de Porto Alegre, entre os anos 2002 e 2005, quando se avaliou o vW nestes 50 casos e em 53 controles. Decorridos42 meses da última inclusão, todas pacientes com CM foram chamadas para nova avaliação do vW por imunoturbidimetria. Consideramos valores elevados de vW quando acima de 160%. As pacientes foram analisadas de acordo com faixa etária de até 59 anos ou ≥ 60 anos de idade. O desfecho clínico foi dividido entre bom (sem evidência de doença) e ruim(óbito ou com evidência de doença). Resultados: Obtivemos nova coleta em 41 pacientes que retornaram para revisão. O fator vW isoladamente não apresentou um bom desempenho prognóstico para o seguimento do CM em relação ao desfecho clínico. Presença de linfonodos axilares positivos, índice de massa corporal (IMC) ≥30 kg/m2, menarca ≤11 anos e tamanho tumoral >2 cm foram as variáveis significativas para determinação do prognóstico nesta coorte. Conclusões: Neste estudo o fator de vW não se mostrou útil para determinação de prognóstico no CM, enquanto que o estado axilar e o diâmetro tumoral foram fatores determinantes de sobrevida. Mais estudos são necessários para confirmar se a menarca e o IMC também são fatores prognósticos.

Background: The Von Willebrand factor (vW) is a glycoprotein whose high serum levels are associated with the mechanism of endothelium stimulation promoting metastatic processes. Aim: this study is to assess its prognostic value in breast cancer, as well as other clinic variables. Methods: We evaluated a cohort of 50 patients with breast cancer (BC) who were being treated at the Mastology Department of Hospital de ClМnicas de Porto Alegre from 2002 to 2005. We measured the vW levels of these 50 patients and 53 controls. Forty-two months after the last patient was included in the study, all patients with BC had their vW levels measured by means of immunoturbidimetric assay. vW values above 160% were considered high. The patients were divided into two age groups: 59 years old or younger and 60 years old or older. The clinical outcome was good (no evidence of disease) and poor (death or evidence of disease). Results: Forty-one patients provided new blood samples. Of the nine patients who did not show up for new tests, five died of BC and one died of myocardial infarction with no evidence of BC (excluded from the study). Three patients did not return because they were living in other cities and were free of disease in their last visit. Conclusions: vW factor was not a good predictive factor for BC regarding the outcome suggested; however, the other variables analyzed, such as presence of positive axillary lymph nodes, BMI 930, menarche =11 years old, and tumor size >2 cm, were significant for the prognostic of this cohort. Conclusions: In this study vW as not useful to determine prognosis in breast cancer, while axilary status and tumoral sizewere predictive factors of survival. More studies are necessary to confirm if menarche and BMI are prognostic factors too.

Rosuvastatin and vascular dysfunction markers in pulmonary arterial hypertension: a placebo-controlled study

We investigated whether chronic rosuvastatin administration could improve the abnormalities of the circulating levels of vascular dysfunction markers in pulmonary arterial hypertension (PAH). Sixty patients, aged 13 to 60 years, with idiopathic (N = 14) or congenital heart disease-associated PAH (N = 46) were equally but randomly assigned to rosuvastatin treatment (10 mg a day, orally) or placebo for 6 months in a blind fashion. Plasma levels of P-selectin, tissue-plasminogen activator and its inhibitor as well as von Willebrand factor antigen were measured by enzyme-linked immunoassay before and after 1, 3, and 6 months of treatment. Baseline levels of biomarkers were elevated (68, 16, 45 and 46 percent increase relative to controls, for P-selectin, von Willebrand factor antigen, tissue-plasminogen activator and its inhibitor, respectively; P < 0.001). P-selectin values at baseline, 1, 3, and 6 months were 39.9 ± 18.5, 37.6 ± 14.6, 34.8 ± 14.6, and 35.4 ± 13.9 ng/mL, respectively, for the rosuvastatin group and 45.7 ± 26.8, 48.0 ± 26.9, 48.1 ± 25.7, and 45.7 ± 25.6 ng/mL for the placebo group. The P-selectin level was lower in the rosuvastatin group compared with placebo throughout treatment (P = 0.037, general linear model). A trend was observed towards a decrease in tissue-plasminogen activator in the statin group (16 percent reduction, P = 0.094), with no significant changes in the other markers. Since P-selectin is crucial in inflammation and thrombosis, its reduction by rosuvastatin is potentially relevant in the pathophysiological scenario of PAH.

Thrombotic thrombocytopenic purpura.

A 6-year-old boy presented with microangiopathic hemolytic anemia, thrombo-cytopenia, altered sensorium and intractable bleeding. A diagnosis of thrombotic thrombocytopenic purpura was made and the child recovered dramatically after plasmapheresis. Recent developments in the understanding of TTP are reviewed, including the importance of a metaloprotease required to cleave multimers of von Willibrand factor.

von Willebrand factor antigen levels in plasma of patients with malignant breast disease

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 + or - 78 U/dl in patients with cancer, 148.4 + or - 59 U/dl in patients with benign disease and 130.6 + or - 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 + or - 113 U/dl, stage IIIB = 194.0 + or - 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 + or - 65 U/dl, stage IIA = 146.9 + or - 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression

Preparation of a heterologus antiserum for the determination of von Willebrand factor in human plasma

A simple method for the preparation of rabbit antiserum against human von Willebrand factor (vWF) from commercial lyophilized factor VIII concentrate is described. vWF antigen (vWFAg)-like protein was obtained by gel filtration of the concentrate on Sepharose 4BTM. A combination of measurements of protein content by absorbance at 280 nm, and of vWFAg by electroimmunoassay using a commercial antibody, provided the data needed to select the Sepharosefiltered fractions with the highest concentrations of vWFAg-like protein. The immunization scheme used induced high antibody titers from the 45th to the 126th day after the first immunization. The resulting antiserum showed a performance similar to that of a commercial preparation in terms of vWFAg determination by electroimmunoassay and two-dimensional crossed-immunoelectrophoresis.

Caracterización de 11 anticuerpos monoclonales anti factor von Willebrand / Characterization of 11 anti von Willebrand factor monoclonal antibodies

Se produjeron y caracterizaron 11 anticuerpos monoclonales de hibridomas murinos anti factor von Willebrand humano. Todos mostraron afinidad específica por el factor von Willebrand humano y pertenecen a los isotipos IgG1, IgG2a e IgG2b. Se estudió el efecto de los anticuerpos sobre la actividad Cofactor Ristocetina, encontrándose que 4 de ellos produjeron inhibición parcial