CD77 levels over enzyme replacement treatment in Fabry Disease Family (V269M) / Níveis de CD77 ao longo do tratamento de reposição enzimática em família com Doença de Fabry (V269M)

ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.

PD-1/PD-L1 regulates Treg differentiation in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo / Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFN ? , IL-2 y TNFa frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi . Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFN ? , IL-2 y TNFa fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi . Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica.

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.

Relationship of macrophage migration inhibitory factor levels in PBMCs, lesional skin and serum with disease severity and activity in vitiligo vulgaris

Melanocyte loss in vitiligo vulgaris is believed to be an autoimmune process. Macrophage migration inhibitory factor (MIF) is involved in many autoimmune skin diseases. We determined the possible role of MIF in the pathogenesis of vitiligo vulgaris, and describe the relationship between MIF expressions and disease severity and activity. Serum MIF concentrations and mRNA levels in PBMCs were measured in 44 vitiligo vulgaris patients and 32 normal controls, using ELISA and real-time RT-PCR. Skin biopsies from 15 patients and 6 controls were analyzed by real-time RT-PCR. Values are reported as median (25th-75th percentile). Serum MIF concentrations were significantly increased in patients [35.81 (10.98-43.66) ng/mL] compared to controls [7.69 (6.01-9.03) ng/mL]. MIF mRNA levels were significantly higher in PBMCs from patients [7.17 (3.59-8.87)] than controls [1.67 (1.23-2.42)]. There was also a significant difference in MIF mRNA levels in PBMCs between progressive and stable patients [7.86 (5.85-9.13) vs 4.33 (2.23-8.39)] and in serum MIF concentrations [40.47 (27.71-46.79) vs 26.80 (10.55-36.07) ng/mL]. In addition, the vitiligo area severity index scores of patients correlated positively with changes of both serum MIF concentrations (r = 0.488) and MIF mRNA levels in PBMCs (r = 0.426). MIF mRNA levels were significantly higher in lesional than in normal skin [2.43 (2.13-7.59) vs 1.18 (0.94-1.83)] and in patients in the progressive stage than in the stable stage [7.52 (2.43-8.84) vs 2.13 (1.98-2.64)]. These correlations suggest that MIF participates in the pathogenesis of vitiligo vulgaris and may be useful as an index of disease severity and activity.

O impacto da fibrose cística no perfil imunológico de pacientes pediátricos / The impact of cystic fibrosis on the immunologic profile of pediatric patients

OBJETIVO: Comparar o estado imunológico de 44 pacientes pediátricos com fibrose cística (FC)a umgrupo-controle formado por 16 indivíduos saudáveis. MÉTODOS: Foram selecionados para o estudo pacientes com FC com idade entre 3 e 12 anos, apresentando um escore clínico moderado e bom. Foram avaliados a glutationa eritrocitária, a produção de espécies reativas de oxigênio, citocinas (TNF-α, IFN-γ, IL-8, IL-6, IL-10) em culturas de células mononucleares do sangue periférico em condições espontâneas e estimuladas por BCG ou PHA, a concentração sérica de TGF-β2, IgA, IgG, IgM, IgE e IgA salivar. RESULTADOS :A produção espontânea de TNF-α, IL-6 e IL-10, a produção de IL-6 estimulada por PHA e TGF-β2, IgA e IgG séricas aumentaram em amostras de pacientes com FC. Indivíduos saudáveis tiveram uma produção mais elevada de TNF-α em resposta a BCG. CONCLUSÃO: Apesar de os pacientes com FC parecerem clinicamente estáveis, os resultados de seus exames de sangue periférico mostraram que houve um impacto sobre o sistema imunológico.

OBJECTIVE: To compare the immunologic state of 44 pediatric patients with cystic fibrosis (CF) with a control group consisting of 16 healthy individuals. METHODS: CF patients aged 3 to 12 years with moderate to good clinical score were selected for the study. Erythrocytic glutathione, production of reactive oxygen species, cytokines (TNF-α, IFN-γ, IL-8, IL-6, IL-10) in peripheral blood mononuclear cells cultures under spontaneous and BCG- or PHA-stimulated conditions, serum concentrations of TGF-β2, IgA, IgG, IgM, IgE, and salivary IgA were evaluated. RESULTS: The spontaneous production of TNF-α, IL-6, and IL-10, the PHA-stimulated production of IL-6, and the serum TGF-β2, IgA, and IgG were increased in samples from CF patients. Healthy subjects had a higher production of TNF-α in response to BCG. CONCLUSION: Although CF patients appearedclinically stable, the results of their peripheral blood examinations demonstrated an impact on the immune system.

Determinación de cobre, magnesio y zinc en leucocitos mononucleares mediante espectrometría de absorción atómica con llama / Determination of copper, magnesium and zinc in mononuclear leukocytes by flame atomic absorption spectrometry

En este trabajo se evaluó un nuevo microinyector de inyección en flujo para la determinación de Cu, Mg y Zn en células mononucleares de la sangre. Este dispositivo permitió analizar muestras en el orden de microlitros mediante espectrometría de absorción atómica con llama, es fácil de construir y adaptar al inyector convencional del espectrofotómetro de absorción atómica. En la determinación de Cu, Mg y Zn se obtuvieron límites de detección de 106, 65 y 37 µg L-1,respectivamente. En las pruebas de recuperación de estos elementos en leucocitos mononucleares se encontraron porcentajes de recuperación entre 98 y 110%.

In this paper we evaluated a new micro-flow injector for the determination of the concentrations of Cu, Mg and Zn in mononuclear blood cells. This device analyzed sample volumes in the order of microliters by flame atomic absorption spectrometry; it is inexpensive, and easy to build and to adapt to the conventional injector of the atomic absorption spectrophotometer. Detection limits of 106, 65 and 37 µg L-1 for Cu, Mg and Zn were obtained, respectively. The percentages of recovery tests were found between 98 and 110%.

Absolute measurement of androgen receptor mRNA in peripheral blood mononuclear, preputial skin and urethral mucosa cells of control individuals with phimosis using qRT-PCR / Valor absoluto do mRNA do receptor androgênico em células mononucleares do sangue periférico e células da pele do prepúcio e mucosa uretral em indivíduos controle com fimose, determinado por meio de qRT-PCR

INTRODUCTION: Androgen actions are exerted upon the androgen receptor (AR), and complete genital virilization of normal 46,XY individuals depends on adequate function and expression of the AR gene in a tissue-specific manner. OBJECTIVE: Standardization of normal ARmRNA in androgen-sensitive tissues. MATERIALS AND METHODS: In this study, we determined the quantitative amounts of ARmRNA in peripheral blood mononuclear, urethral mucosa and preputial skin cells of control subjects with phimosis by using RT-PCR. RESULTS: The mean (SD) values of AR expression in blood, urethra and prepuce were: 0.01 (0.01); 0.43 (0.32); 0.31 (0.36), respectively. CONCLUSION: The AR expression is low in blood and equivalent in urethral mucosa and preputial skin, which may be useful in the diagnosis of individuals with abnormal external genitalia.

INTRODUÇÃO: As ações androgênicas são exercidas por meio do receptor androgênico (AR), e a completa virilização genital de indivíduos 46,XY normais depende de adequada expressão do gene AR de forma tecido específica. OBJETIVO: Padronizar valores normais de ARmRNA em tecidos sensíveis aos andrógenos. MATERIAIS E MÉTODOS: Neste estudo, determinamos as quantidades de ARmRNA em células mononucleares do sangue periférico e em células da mucosa uretral e pele do prepúcio de indivíduos controles com fimose, utilizando RT-PCR. RESULTADOS: A média (dp) dos valores de expressão do AR em sangue, uretra e prepúcio foram: 0,01 (0,01); 0,43 (0,32); 0,31 (0,36), respectivamente. CONCLUSÃO: A expressão do AR é baixa em sangue periférico e equivalente em mucosa uretral e pele prepucial, sendo sua quantificação útil no diagnóstico de indivíduos com alterações da genitália externa.

Leukemic phase of anaplastic lymphoma kinase positive, anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a distinct type of CD30+ T/null-cell non-Hodgkin's lymphoma that frequently involves nodal and extranodal sites. The presence of leukemic phase in ALCL is extremely rare and occurs exclusively with ALK1-positive ALCL. We describe two patients with ALK1-positive ALCL who developed a leukemic phase with rapid progression of the disease. Immunophenotypic pattern assessed on peripheral blood by flow cytometry revealed CD45, CD30, and CD25 positivity in both cases but NPM-ALK1 was expressed in only one case. Both patients developed leukemic phase as a terminal event of the disease and we share the immunophenotypic features of both cases.

Clinical and immunological features of patients with atopy and concomitant HTLV-1 infection

Human T-cell lymphotropic virus type 1 (HTLV-1) induces an exacerbated type 1 immune response characterized by high spontaneous IFN-γ and TNF-α production. Allergic rhinitis and asthma are associated with the type 2 immune response, with elevated secretion of IL-4 and IL-5. The aim of this study was to characterize the immune response in atopic HTLV-1 carriers. The cytokine profile of atopic HTLV-1 carriers (N = 10; all females) was compared with that of non-atopic HTLV-1 carriers (N = 14; 9 females and 5 males). Mean patient age of atopic and non-atopic groups was 45 ± 8 and 38 ± 11 years, respectively. All atopic HTLV-1 carriers had rhinitis with or without asthma and a skin prick test positive for Dermatophagoides pteronyssinus antigen 1 (Derp-1). There was no difference in cytokine levels between the two groups in unstimulated peripheral blood mononuclear cell cultures. In cultures stimulated with Derp-1, IFN-γ levels tended to be higher (P = 0.06) and IL-5 levels were higher (P = 0.02) in atopic HTLV-1 patients than in non-atopic subjects. In contrast, IL-10 was lower (P = 0.004) in atopic than in non-atopic HTLV-1-infected subjects. This study shows that HTLV-1 infection with an exaggerated type 1 immune response does not prevent atopy. In this case, the exacerbated type 1 and type 2 immune responses were due to a lack of IL-10 production, a cytokine that plays an important role in down-modulating type 1 and type 2 immune responses and in preventing the development of chronic inflammatory diseases.

Aislamiento de células mononucleares de sangre periférica para trasplante de células madre: método simplificado / Isolation of mononuclear cells of peripheral blood for stem cell transplant: simplified method

En los últimos años el tema de las células madre ha despertado creciente interés por su potencial terapéutico en enfermedades que hasta el momento no tienen un tratamiento efectivo. Se realizó un estudio prospectivo y exploratorio en pacientes con arteriosclerosis obliterante de miembros inferiores, en el que se evaluó la seguridad y efectividad de un método manual de recolección y procesamiento de células mononucleares y de células CD34+ a partir de sangre periférica movilizada. La sangre se procesó en sistemas cerrados de recolección, utilizando el hidroxietilalmidón como potenciador de la sedimentación eritrocitaria. Los pacientes fueron tratados con factor estimulador de colonias granulocítico en dosis total de 40ìcg/kg de peso durante 2 días, y después si el conteo de leucocitos era superior a 20 x 109/L se procedió a la autodonación. Para valorar la eficacia del método se analizaron las cantidades de células nucleadas, de células mononucleares y de células CD 34+ en el concentrado celular; se determinó la viabilidad celular y además se hizo el estudio microbiológico del material obtenido. Se demostró que el método es eficaz y seguro, ya que logra niveles celulares adecuados, con elevada viabilidad y ausencia de contaminación bacteriana. Por otra parte, es sencillo y de bajo costo, lo que permite su extensión a otros centros de salud, en particular a los de menos recursos. Esto facilita que un mayor número de pacientes se puedan beneficiar con el tratamiento a base de células madre.

In the last years, the topic of stem cells has arisen an increasing interest for its therapeutic potential in diseases that have not an effective treatment so far. A prospsective and exploratory study was conducted in patients with obliterant atherosclerosis of the lower limbs to evaluate the safety and effectiveness of a manual method of collection and processing of mononuclear cells and of CD34+ cells, starting from mobilized peripheral blood. The blood was processed in closed collection systems, using hydroxyethyl starch as a potentiator of erythrocyte sedimentation. The patients were treated with granulocyte colony stimulating factor at total doses of 40µcg/kg of weight during 2 days. Self-donation was performed when the leukocyte count was higher than 20 x 109/L. To assess the efficacy of the method, the amounts of nucleated cells, of mononuclear cells and of CD 34+ cells in the cellular concentrate were analyzed. Cellular viability was determined and a microbiological study of the material obtained was conducted. It was proved that the method is efficient and safe, since adequate cellular levels with a high viability and absence of bacterial contamination are attained. On the other hand, it is simple and cheap, which allows its application in other health centres, particularly in those with less resources. This makes possible that more patients benefit from the stem cell treatment.

Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [³H]-quinuclidinylbenzilate ([³H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means ± SEM; control: 58.69 ± 5.54, N = 29; Chagas: 72.29 ± 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 ± 2.45, N = 18; Chagas: 20.22 ± 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 ± 3.83; Chagas: 43.62 ± 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 ± 3.84, N = 4; Chagas: 54.38 ± 6.28 fmol/mg, N = 20); 2) [³H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 ± 2321; control 10,940 ± 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1â, was increased in both plasma of chagasic rats and in the culture medium, and TNF-á level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

Paracoccidioidomycosis: no genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Paracoccidioidomicose é micose sistêmica causada pelo Paracoccidioides brasiliensis. Considerando que doenças infecciosas são capazes de induzir danos genéticos, o objetivou-se analisar quebras no DNA em células de sangue periférico de pacientes com paracoccidioidomicose utilizando o teste do cometa. Os resultados sugeriram que essa enfermidade não exerce genotoxicidade.

Spontaneous neutrophil activation in HTLV-1 infected patients

Human T cell lymphotropic Virus type-1 (HTLV-1) induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT) to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy); 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS)-stimulated neutrophil activity (reduction of NBT to formazan). The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p<0.001) in HTLV-1 infected subjects than in healthy individuals. A trend towards a positive correlation was noted, with increasing percentage of NBT positive neutrophils and levels of IFN-gamma. The high IFN-gamma producing HTLV-1 patient group had significantly greater NBT than healthy controls, 43±24 percent and 17±4.8 percent respectively (p< 0.001), while no significant difference was observed between healthy controls and the low IFN-gamma-producing HTLV-1 patient group (30±20 percent). Spontaneous neutrophil activation is another marker of immune perturbation resulting from HTLV-1 infection. In vivo activation of neutrophils observed in HTLV-1 infected subjects is likely to be the same process that causes spontaneous IFN-gamma production, or it may partially result from direct IFN-gamma stimulation.

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Expression of HLA-DR antigen in large bowel carcinoma

One hundred large bowel carcinomas were studied immunohistochemically with regard to expression of HLA-DR antigen (DR). One or two sections from each tumor including surrounding normal mucosa were examined by a semiquantitative counting system for tumor cells and mucosal and stromal infiltrates of lymphocytes and mononuclear cells (MNCs) with DR expression and the results were applied Chi-square test. The rate of presence of DR positive (DR+) lymphocytes in lymphoid nodules and DR+ lymphocytes/ MNC in the adjacent mucosa and stroma in DR+ carcinoma (50%) was higher (P < 0.01) than in DR- carcinoma (21.9%). Thirty-six carcinomas (36%) were DR+. Three (75%) out of 4 DR+ poorly differentiated carcinomas and six (20%) out of 30 DR+ moderately differentiated carcinomas showed homogeneously strong DR+ expression. There was tendency for poorly differentiated carcinoma to be more homogeneous DR+ expression. According to Dukes' stage, four (80%) out of 5 carcinomas in Dukes' stage D were DR-. An increased infiltration of lymphocytes/MNCs into adjacent mucosa and stroma in large bowel carcinomas is possibly related with DR expression by carcinoma. From the results of this study, we postulated as follows: 1) DR+ tumor cells may act as antigen-presenting cells, 2) They may have an inhibitory effect for distant metastasis, 3) Poorly differentiated carcinoma expressed more DR+ homogeneously.

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax

The cellular immune response to the circumsporozoite (CS) protein of plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequenceof the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen specific in vitro proliferative responseto the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative reponse when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, withinthe major surface antigen of P. vivax sporozoites, of epitopes capble to induce proliferation of human PBMC

Magnésio mononuclear em hipertensos idosos tratados com tiazídico / Mononuclear magnesium in aged hypertensive patients treated with thiazide

Purpose­To study mononuclear magnesium and serum cations (Na, K and Mg) in elderly hypertensive patients treated with hydrochlorothiazide during 90 days. Patients and Methods­ 15 elderly hypertensive patients treated with hydrochlorothiazide, 25 mg/day or placebo. A method of freezing produced total Iysis of the cells; Mg was measured by atomic spectrophotometry. Results­No differences were noted in mononuclear or serum magnesium in the thiazide or placebo treated patients, and the diuretic produced significant decreases in supine systolic and diastolic blood pressure. Conclusion­Magnesium supplementation does not seem essencial in most patients in this conditions