Lifewide profile of cytokine production by innate and adaptive immune cells from Brazilian individuals.

BACKGROUND: Immunosenescence is associated with several changes in adaptive and innate immune cells. Altered cytokine production is among the most prominent of these changes. The impact of age-related alterations on cytokine global profiles produced by distinct populations of leukocytes from healthy Brazilian individuals was studied. We analysed frequencies of cytokine-producing lymphocytes and innate immune cells from individuals at several ages spanning a lifetime period (0-85 years). RESULTS: Healthy adult individuals presented a balanced profile suggestive of a mature immune system with equal contributions of both innate and adaptive immunity and of both categories of cytokines (inflammatory and regulatory). In healthy newborns and elderly, innate immune cells, especially neutrophils and NK-cells, contributed the most to a balanced profile of cytokines. CONCLUSIONS: Our results support the hypothesis that ageing is not associated with a progressive pro-inflammatory cytokine production by all leukocytes but rather with distinct fluctuations in the frequency of cytokine-producing cells throughout life.

The in vitro exposure to cypermethrin does not inhibit the proliferative response of peripheral blood mononuclear cells.

This study evaluated the effect of in vitro exposure to cypermethrin on peripheral blood mononuclear cells proliferative response, considering reduced peripheral blood mononuclear cells proliferative response observed in individuals occupationally exposed to pyrethroids. Peripheral blood mononuclear cells were obtained from 21 healthy subjects (28.0 ± 9.0 years old). The effect of cypermethrin (at 0.5, 1.0 and 5.0 mg/ml) on cell viability was evaluated by flow cytometry using an apoptosis detection kit. Cell proliferation (PI) was evaluated by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence decay using flow cytometry. Cells labeled with CFSE were exposed, in vitro, to cypermethrin (0.5, 1.0, 2.0, 2.5 and 4 µg/ml) and stimulated with phytohemagglutinin (PHA 1.0 or 5.0 µg/ml) for 5 d (37 °C, 5% CO2). The in vitro treatment of peripheral blood mononuclear cells with cypermethrin did not induce apoptosis or necrosis after 5 d in culture. Stimulation by PHA induced cell proliferation (PI = 1.29 ± 1.09 and 2.01 ± 0.62, PHA at 1.0 and 5.0 µg/ml, respectively, mean ± SD) and in vitro exposure to cypermethrin did not alter cellular proliferative response to PHA (PI = 1.80 ± 0.50, 2.60 ± 0.05 and 2.10 ± 1.20 for cypermethrin at 1.0, 2.0 and 4.0 µg/ml, respectively, and PHA at 5.0 µg/ml). In vitro treatment of peripheral blood mononuclear cells with cypermethrin, at the doses tested, does not affect cell viability or proliferation. These findings suggest that the reduction of proliferation observed on lymphocytes derived from individuals occupationally exposed to pesticides may be related to other mechanisms than direct action of cypermethrin on lymphocytes.

The experimental model of nephrotic syndrome induced by Doxorubicin in rodents: an update.

Nephrotic syndrome (NS) is characterized by proteinuria, hypoalbuminemia, generalized edema, and hyperlipidemia. It begins by changes in the glomerular filtration barrier, with increased permeability to plasma proteins. It affects all age groups and can progress to end-stage renal disease. NS pathophysiology is still unknown. However, the critical role of the immune system is well recognized. Animal models are useful tools for the investigation of NS. Among different experimental models proposed in the literature, disease induced by Doxorubicin has been considered helpful to the purpose of many studies. The aim of this review article is to describe the animal model of NS induced by the injection of Doxorubicin in rodents, with emphasis on action of the drug, potential mechanisms of renal injury, as well biochemical, histological, and corporal changes obtained with this model.

Increased Migratory and Activation Cell Markers of Peripheral Blood Lymphocytes in an Experimental Model of Nephrotic Syndrome.

The present study aimed to evaluate the expression of CD80 and CD18 in subpopulations of peripheral blood leukocytes and oxidative kidney damage in rats with nephrotic syndrome (NS) induced by doxorubicin (Dox) in comparison to control animals at different time points. Male adult Wistar rats were submitted to 24-hour urine and blood collection for biochemical and immunological analysis at 7, 14, 21, and 28 days after Dox injection. After euthanasia, the kidneys were removed for histological analysis and the evaluation of oxidative stress. The phenotypic characterization of leukocytes was performed using flow cytometry. Dox-injected animals exhibited increased CD18 expression in cytotoxic T lymphocytes, NK cells, and monocytes and high CD80 expression in monocytes. Kidney oxidative damage was positively correlated with CD80 expression in monocytes and serum levels of creatinine. These results suggest that phagocytic and cytotoxic cells are preferentially recruited to the tissue injury site, which may contribute to kidney dysfunction in this animal model of NS. The blockade of integrin and costimulatory molecules may provide new therapeutic opportunities for NS.

The role of the immune system in idiopathic nephrotic syndrome: a review of clinical and experimental studies.

Idiopathic nephrotic syndrome (INS) is a multifactorial disease, characterized by proteinuria, hypoalbuminemia, edema and hyperlipidemia. Studies in humans and animal models have associated INS with changes in the immune response. The purpose of this article is to review clinical and experimental findings showing the involvement of the immune response in the pathogenesis of INS. The role of the immune system in INS has been shown by clinical and experimental studies. However, the pattern of immune response in patients with INS is still not clearly defined. Many studies show changes in the dynamics of T lymphocytes, especially the regulatory T cells. Alternatively, there are other reports regarding the involvement of the complement system and B lymphocytes in the pathophysiology of INS. Indeed, none of the immunological biomarkers evaluated were undeniably linked to changes in glomerular permeability and proteinuria. On the other hand, some studies suggest a link between urinary chemokines, such as IL-8/CXCL8 and MCP-1/CCL2, and changes in glomerular permeability and/or the deterioration of glomerulopathies. To understand the pathophysiology of INS, longitudinal studies are clearly needed. The characterization of the profile of the immune response might help the development of specific and individualized therapies, leading to clinical improvement and better prognosis.

Effect of essential oil from Ageratum fastigiatum on beta-integrin (CD18) expression on human lymphocytes stimulated with phorbol myristate acetate in vitro.

Agareratum fastigiatum is a Brazilian medicinal plant used as anti-inflammaroty and for wound healing by the folk medicine. In vitro and in vivo studies involving A. fastigiatum essential oil (EOAF) showed indications of anti-inflammatory activity, however, its effect on membrane integrins involved on cell migration is still unclear. Hence, it was evaluated in the present study the effect of EOAF on CD18 frequency on human lymphocytes. By using gas chromatography/mass spectrometry it was identified 9 compounds on EOAF: α-pinene; ß-pinene; ß-myrcene; d-limonene; ß-ocimene; sesquiterpenes; α-copaene; 4,8-ß-epóxi-caryophyllene; germacrene and bicyclogermacrene. On in vitro tests, 6.25 × 10-3 and 12.5 × 10-3 µL/mL EOAF reduced CD18 frequency on phorbol-12-myristate-13-acetate (PMA)-stimulated lymphocytes. Such cells were obtained from peripheral blood of healthy volunteers, and were treated or not with EOAF. They were stained with fluorescent anti-CD18 monoclonal antibodies, after 24 hours incubation. Our data corroborates previous findings, indicating a possible anti-inflammatory activity of EOAF.

Pediatric Patients With Steroid-Sensitive Nephrotic Syndrome Have Higher Expression of T Regulatory Lymphocytes in Comparison to Steroid-Resistant Disease.

Background and Aim: Idiopathic nephrotic syndrome (INS) is classified according to the response to drug therapy in steroid-sensitive (SS), steroid-dependent (SD), and steroid-resistant (SR) categories. Previous studies showed changes in inflammatory activity of subpopulations of lymphocytes in INS. This study aimed to compare SS and SR patients in regard to subpopulations of leukocytes, profile of regulatory lymphocytes, and migratory activity of lymphocyte subpopulations. Results obtained in INS patients were also compared to age and sex-matched healthy controls. Methods: This is a cross-sectional study including SS patients (n = 30), SR patients (n = 14), and controls (n = 10). Peripheral blood samples were withdrawn for ex-vivo leukocyte flow cytometry analysis. Results: Percentage of B-lymphocytes and natural killer (NK) cells were significantly reduced in SR patients when compared to controls, while the percentage of NKT cells were decreased in SS patients in comparison to controls. Percentages of CD4+ expressing FoxP3 and CTLA4 were significantly higher in SS patients in comparison to SR patients and controls. The expression of integrin CD18 on the surface of T lymphocytes (CD3+) was reduced in SS patients if compared to controls. Conclusion: This study found that SS INS patients have higher levels of regulatory T-lymphocytes and lower expression of adhesion molecules than SR patients.

Background autofluorescence induced by plant extracts in human lymphocytes: A flow cytometric analysis of a critical bias.

The presence of background autofluorescence sources is considered as an important problem when performing fluorometric methods, due to the possible spectral overlap between it and the fluorescence emission of probes. Regarding that, we evaluated the presence of background autofluorescence in human lymphocytes after the treatment with extracts from three medicinal plants, including ethanolic extract from aerial parts of Ageratum fastigiatum, ethanolic extract from aerial parts of Eriosema campestre and the ethanolic extract from stem of Pseudobrickellia brasiliensis. Human peripheral blood mononuclear cells were treated with each extract in vitro during 24 h, followed by flow cytometric analysis. Additionally, the fluorescence emission of plant extracts was evaluated by fluorometry, using the same concentrations used in cell cultures. We identified that plant extracts treatment on lymphocytes induced background autofluorescence detectable in several wavelength ranges. Isolated extracts showed no expressive fluorescence emission in fluorometric analyses, suggesting that background autofluorescence was induced in lymphocytes by interactions between cellular components and extracts compounds. Here we discuss the importance to perform previous tests to evaluate a possible background autofluorescence induction after cell treatments with plant extracts or any other substance. In spite of being mandatory, background autofluorescence analysis of cells after treatments and stimulations is still underestimated on literature. In summary, following the precautions herein established should help to reduce the incidence of false positive results.

Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats.

Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5 mg/kg of DXR and were sacrificed at seven, 14, 21 and 28 days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII-XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II-III and VIII) at seven and 14 days. At 21 and 28 days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.

[Evaluation of the performance of immunological parameters as indicators for clinical progression of chronic HTLV-1 infection]. / Avaliação do desempenho de parâmetros imunológicos como indicadores de progressão clínica da infecção crônica pelo HTLV-1.

This study evaluated the performance of single and combined laboratory parameters, B-lymphocyte percentages (%LB), T/B cell ratio and %CD8+HLA-DR+/CD8+, to differentiate asymptomatic cases (AS) from HAM/TSP patients (HT) within a population of HTLV-1 seropositive cases. Percentage indices demonstrated that each parameter alone presented moderate performance, with co-negativity of 83 and 91% for %LB and T/B cell ratio, respectively, and co-positivity of 78% for %CD8+HLA-DR+/CD8+. Combined analysis (%CD8+HLA-DR+/CD8+ and T/B cell ratio) did not show any substantial performance enhancement (co-positivity = 75% and co-negativity = 74%). Likelihood ratio analysis using different value ranges for the separate parameters revealed that HTLV-1 seropositive cases with %LB<7%, T/B cell ratio>11 and %CD8+HLA-DR+/CD8+>70% would have, respectively, 11, 19 and 10 times greater chance of belonging to the HT group. Therefore, use of these phenotypic indicators as complementary laboratory methods for monitoring the clinical progression of chronic HTLV-1 infection is recommended.

Are lipid disorders involved in the predominance of human T-lymphotropic virus-1 infections in women?

INTRODUCTION: The human T-lymphotropic virus-1 (HTLV-1) is associated with chronic inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease. Disturbances in lipid metabolism are involved in inflammatory and demyelinating diseases. METHODS: Plasma levels of triglycerides, total cholesterol, and fractions of HTLV-1-infected individuals of both sexes with different clinical progressions were determined. RESULTS: Elevated levels of triglyceride and very low-density lipoproteins (VLDL) were exclusively detected in HTLV-1-infected women from asymptomatic and HAM/TSP groups compared with uninfected individuals (p = 0.02). CONCLUSIONS: Elevated triglyceride and VLDL levels in HTLV-1-infected women may be related to the predominance of HAM/TSP in women.

Effect of aerobic training on plasma cytokines and soluble receptors in elderly women with knee osteoarthritis, in response to acute exercise.

The aim of this study was to evaluate levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and soluble forms of the TNF-α receptor (sTNFR1 and sTNFR2) from plasma taken from the peripheral blood of elderly individuals presenting with osteoarthritis (OA) of the knee. These patients underwent aerobic treatment through the use of physical exercises. The study consisted of a longitudinal analysis of older individuals presenting clinical and radiographic diagnosis of knee OA that were submitted to 12 weeks of aerobic treatment. The individuals were evaluated during acute exercise or after chronic exercise. During acute exercise (walking slowly on the mat), blood samples of the patients were collected before, immediately after, and 30 min following the end of training. After chronic exercise (aerobic walking training, three times/week for 12 weeks), patient blood samples were obtained for comparison. Additionally, clinical and functional assessments (WOMAC test and 6-min walk) were performed at the end of all physical exercises. Plasma concentrations of cytokines and soluble receptors were measured by ELISA. Aerobic training increased the plasma concentration of sTNR1; however, it decreased the plasma concentration of sTNFR2, when compared with levels of resting patients. Acute exercise differentially affects the levels of sTNFR1 dependent on when the samples were taken, before and after aerobic training. However, the levels of sTNFR2 were not affected by training. For the population studied, we observed differences in the levels of sTNFR1 and sTNFR2 following acute and chronic exercise. Other additional factors, like the level of inactivity of the individual and the type of physical exercise that patients are exposed to, need to be considered as well. The variation in the levels of soluble receptors correlated with functional improvement; however, the inflammatory osteoarthritis markers (IL-6 and TNF-α) were unaffected by the walking exercises.

Performance of IgG and IgG1 anti-HTLV-1 reactivity by an indirect immunofluorescence flow cytometric assay for the identification of persons infected with HTLV-1, asymptomatic carriers and patients with myelopathy.

In this study, the performance of IgG and IgG1 anti-HTLV-1 reactivity obtained by a flow cytometric assay was evaluated to verify its applicability for the diagnosis of persons infected with HTLV-1, including asymptomatic carriers and patients with myelopathy. The ability to identify patients with myelopathy among persons infected with HTLV-1 was also examined. Western blot assays were performed to assess the reactivity profiles of sera from asymptomatic carriers and patients with myelopathy against viral proteins. The data showed that IgG1 detected by flow cytometric assay is effective for the diagnosis of persons infected with HTLV-1 with 97% sensitivity and 100% specificity. IgG and IgG1 exhibited high performance in distinguishing patients with myelopathy from asymptomatic carriers. Using serum dilutions and cut-off points established previously a second HTLV-1 carrier group was tested using flow cytometric assay to detect IgG and IgG1. The data demonstrated sensitivity of 93% and 98%, respectively, confirming the high reactivity of persons infected with HTLV-1 detected by this method. Western blot assays confirmed the high specificity of MT-2 cells as a reliable source of viral antigen since only sera from persons infected with HTLV-1 recognised MT-2 proteins. Furthermore, a high reactivity to Gag and Env proteins was observed, especially among patients with myelopathy. These data suggest that flow cytometric detection of IgG1 is a valuable, non-conventional serological method to diagnose HTLV-1 infection and for research purposes.

Thrombospondin-1 expression in human T-lymphotropic virus 1 asymptomatic carriers and patients with HTLV-associated myelopathy/tropical spastic paraparesis / Expressão de Trombospondina-1 em indivíduos infectados pelo HTLV-1 assintomáticos e pacientes portadores da mielopatia associada ao HTLV/Paraparesia Espástica Tropical

O Vírus Linfotrópico de células T humanas tipo 1 (HTLV-1) está associado a uma mielopatia (chamada mielopatia associada ao HTLV - HAM/TSP). A trombospondina-1 (TSP-1) é uma proteína da matriz que interfere com a adesão, a motilidade, e a proliferação celular. Níveis deexpressão de RNA mensageiro (mRNA) da trombospondina-1 foram avaliados em indivíduos infectados por HTLV-1: 11 pacientes assintomáticos, 18 com mielopatia ou oligossintomáticos, e 13participantes não-infectados. O RNA de células mononucleares do sangue periférico foi submetido à análise de RT-PCR para trombospondina-1. O número de indivíduos que expressaram esta proteína foi maior no grupo com mielopatia/sintomas (14/18, p igual 0,007). Em geral, a tendência para valores mais elevados de mRNA de trombospondina-1 foi observada no grupo de infectados pelo vírus (p igual 0,062). Os níveis mais elevados de expressão do mRNA foram detectados no início dos sintomas clínicos da HAM/TSP. Estudos adicionais com maior número de amostras são necessários para elucidar melhor o papel desta proteína da matriz na rede inflamatória relacionada à HAM/TSP.

Avaliação do desempenho de parâmetros imunológicos como indicadores de progressão clínica da infecção crônica pelo HTLV-1 / Evaluation of the performance of immunological parameters as indicators for clinical progression of chronic HTLV-1 infection

Neste estudo, foi avaliado o desempenho isolado e combinado de parâmetros laboratoriais, percentual de linfócitos B ( por centoLB), a razão entre células T/B e o por centoCD8+HLA-DR+/CD8+, na identificação de indivíduos assintomáticos-AS ou portadores de HAM/TSP-HT numa população de casos soropositivos para HTLV-1. índices expressos em porcentagem demonstram que cada parâmetro, isoladamente, apresenta desempenho moderado, com co-negatividade=83 por cento e 91 por cento para por centoLB e razão entre células T/B, respectivamente e co-positividade=78 por cento para por centoCD8+HLA-DR+/CD8+. A análise combinada ( por centoCD8+HLA-DR+/CD8+ e razão T/B) não revelou ganho significativo no desempenho (co-positividade=75 por cento, co-negatividade=74 por cento). A análise das razões de verossimilhança em diferentes faixas de valores, para os parâmetros isolados, revelou que um indivíduo soropositivo para HTLV-1 com por centoLB<7 por cento, razão entre células T/B>11 e por centoCD8+HLA-DR+/CD8+>70 por cento possui, respectivamente, 11, 19 e quase 10 vezes mais chances de pertencer ao grupo HT. Portanto, recomenda-se o uso desses indicadores fenótipos na propedêutica laboratorial complementar de monitoração da progressão clínica da infecção crônica pelo HTLV-1.

This study evaluated the performance of single and combined laboratory parameters, B-lymphocyte percentages ( percentLB), T/B cell ratio and percentCD8+HLA-DR+/CD8+, to differentiate asymptomatic cases (AS) from HAM/TSP patients (HT) within a population of HTLV-1 seropositive cases. Percentage indices demonstrated that each parameter alone presented moderate performance, with co-negativity of 83 and 91 percent for percentLB and T/B cell ratio, respectively, and co-positivity of 78 percent for percentCD8+HLA-DR+/CD8+. Combined analysis ( percentCD8+HLA-DR+/CD8+ and T/B cell ratio) did not show any substantial performance enhancement (co-positivity = 75 percent and co-negativity = 74 percent). Likelihood ratio analysis using different value ranges for the separate parameters revealed that HTLV-1 seropositive cases with percentLB<7 percent, T/B cell ratio>11 and percentCD8+HLA-DR+/CD8+>70 percent would have, respectively, 11, 19 and 10 times greater chance of belonging to the HT group. Therefore, use of these phenotypic indicators as complementary laboratory methods for monitoring the clinical progression of chronic HTLV-1 infection is recommended.