Efeito da exposição a produtos fumígenos sobre a expressão de metalotioneínas durante a artrite experimental e sobre linfócitos T / Effect of exposure to tobacco-related products on metallothionein expression in the experimental arthritis and on T lymphocytes functions

Produtos liberados pela queima do cigarro convencional (CC) estão relacionados com a progressão clínica da artrite reumatoide (AR). Produtos fumígenos não combustíveis surgiram com a premissa de apresentarem menor toxicidade que o CC, dentre os quais está o tabaco aquecido (heat-not-burn tobacco; HNBT). Neste projeto investigamos os efeitos do HNBT sobre eventos envolvidos na AR, focando na sintomatologia, expressão de metalotioneínas (MTs), e na biologia de linfócitos T CD4+ primários e da linhagem Jurkat. Exposições in vivo ao ar, CC ou HNBT foram realizadas 2 vezes ao dia, 1 hora cada (12 CC ou 24 HNBT/hora), nos dias 14-21 da indução da artrite induzida por antígeno (AIA) em camundongos C57Bl/6. Foram realizadas análises dos parâmetros clínico da doenças, histopatologia e imunohistoquímica; quantificação de nicotina e cotinina séricas por cromatografia líquida acoplada a espectrometria de massas (MS). Os efeitos das exposições in vitro sobre linfócitos T foram mensurados por citometria de fluxo e ELISA. A concentração de metais emitidas pelo CC ou HNBT durante as exposições foram mensurados por MS com plasma acoplado. Camundongos expostos ao CC apresentaram intensa inflamação pulmonar, expressões acentuadas de MTs hepáticas e pulmonares e exacerbação dos parâmetros de AIA quando comparados ao grupo expostos ao HNBT. Animais expostos ao CC ou ao HNBT apresentaram redução na celularidade de órgãos linfoides. Somente a exposição in vitro ao CC causou estresse oxidativo e secreção de citocinas inflamatórias, ativação do receptor de hidrocarbonetos arila (AhR) e polarização de células Th17. Diferentemente, exposição ao CC ou ao HNBT provocaram redução da secreção de IL-2 e proliferação de células Jurkat. A exposição de células Jurkat à nicotina mimetizou os efeitos inibitórios da exposição ao HNBT sobre a secreção de IL-2 e proliferação de linfócitos T. O CC liberou maiores concentrações de metais nas câmaras de exposição. Associados, nossos resultados mostram que embora exposições ao HNBT não exacerbem parâmetros inflamatórios de AIA e nem em funções linfócitos T, ambos produtos prejudicam a celularidade de órgãos linfoides e a proliferação e secreção de IL-2 por linfócitos T

Products released by burning conventional cigarettes (CC) are related to the worsening of rheumatoid arthritis (RA). Non-combustible smoking products appeared with the premise of presenting less toxicity than the CC, among which is the heated tobacco (heat-not-burn tobacco; HNBT). Here, we investigate the effects of HNBT on events involved in RA, focusing on symptoms, expression of metallothioneins (MTs), and on the biology of primary CD4+ T lymphocytes and the Jurkat T cell lineage. In vivo exposures to air, CC or HNBT were performed twice a day, 1 hour each (12 CC or 24 HNBT / hour), on days 14-21 of the induction of antigen-induced arthritis (AIA) in C57Bl / 6 mice. Analyzes of the clinical parameters of the AIA, histopathology, and immunohistochemistry were performed; quantification of nicotine and cotinine by liquid chromatography coupled to mass spectrometry (MS). The in vitro effects of exposures on T lymphocytes were measured by flow cytometry and ELISA. The concentration of metals released by the CC or HNBT during the exposures was measured by MS with coupled plasma. Mice exposed to CC showed intense pulmonary inflammation, marked expressions of hepatic and pulmonary MTs, and exacerbation of AIA parameters when compared to the group exposed to HNBT. Animals exposed to CC or HNBT showed a reduction in the cellularity of lymphoid organs. Only in vitro exposure to CC caused oxidative stress and secretion of inflammatory cytokines, activation of the aryl hydrocarbon receptor (AhR), and polarization of Th17 cells. However, exposure to CC or HNBT led to reduced secretion of IL-2 and proliferation of Jurkat cells. The exposure of Jurkat T cells to nicotine mimicked the inhibitory effects of exposure to HNBT on IL-2 secretion and T lymphocyte proliferation. The CC released higher concentrations of metals in the exposure chambers. In association, our results show that although exposures to HNBT do not exacerbate inflammatory parameters of AIA or T lymphocyte functions, both products impair lymphoid organ cell function and the proliferation and secretion of IL-2 by T lymphocytes

Benzoxazole Derivative B-98 Ameliorates Dextran Sulfate Sodium-induced Acute Murine Colitis and the Change of T Cell Profiles in Acute Murine Colitis Model / 대한소화기학회지

BACKGROUND/AIMS: The unique role of enzyme 5-lipoxygenase (5-LO) in the production of leukotrienes makes it a therapeutic target for inflammatory bowel disease (IBD). The aim of this study was to evaluate the effects of B-98, a newly synthesized benzoxazole derivatives and a novel 5-LO inhibitor, in a mouse model of IBD induced by dextran sulfate sodium (DSS). METHODS: C57BL/6 mice were randomly assigned to four groups: normal control, DSS colitis (DSS+saline), low dose B-98 (DSS+B-98 20 mg/kg) and high dose B-98 (DSS+B-98 100 mg/kg). B-98 was administered with 3% DSS intraperitoneally. The severity of the colitis was assessed via the disease activity index (DAI), colon length, and histopathologic grading. The production of inflammatory cytokines interleukin (IL)-6 was determined by RT-PCR. Th cells were examined for the proportion of Th1 cell, Th2 cell, Th9 cell, Th17 cell and Treg cell using intracellular cytometry. RESULTS: The B-98 group showed lower DAI, less shortening of the colon length and lower histopathologic grading compared with the DSS colitis group (p<0.01). The expression of IL-6 in colonic tissue was significantly lower in the B-98 groups than the DSS colitis group (p<0.05). The cellular profiles revealed that the Th1, Th9 and Th17 cells were increased in the DSS colitis group compared to the B-98 group (p<0.05). CONCLUSIONS: Our results suggest that acute intestinal inflammation is reduced in the group treated with B-98 by Th1, Th9 and Th17 involved cellular immunity.

Immunophenotyping of circulating T cells in a mucosal leishmaniasis patient coinfected with HIV / Immunofenotipagem de células T circulantes em um paciente com leishmaniose mucosa co-infectado com HIV

HIV coinfection modifies the clinical course of leishmaniasis by promoting a Th2 pattern of cytokine production. However, little information is available regarding the lymphocytic response in untreated coinfected patients. This work presents the immunophenotyping of Leishmania-stimulated T cells from a treatment-naÏve HIV+ patient with ML. Leishmania braziliensis antigens induced CD69 expression on CD3+CD4+ and CD3+CD8+ cells. It also increased IL-4 intracellular staining on CD3+CD4+GATA3- population and decreased the percentage of CD3+CD4+IL-17+ cells. This suggests that modulations in the IL-4R/STAT6 pathway and the Th17 population may serve as parasitic evasion mechanisms in HIV/ML. Further studies are required to confirm these results.

A co-infecção por HIV modifica o curso clínico da leishmaniose ao promover aumento no perfil Th2 de produção de citocinas. No entanto, há pouca informação a respeito da resposta linfocitária em pacientes co-infectados sem tratamento. Neste trabalho, foi realizada a imunofenotipagem de células T estimuladas com antígenos de Leishmania braziliensis em paciente não tratado HIV+ e com leishmaniose mucosa. Os resultados mostraram aumento na expressão de CD69 em células CD3+CD4+ e CD3+CD8+. Além disso, foi observado aumento de IL-4 na população de linfócitos CD3+CD4+GATA3- e diminuição no percentual de células CD3+CD4+IL-17+. Estes resultados sugerem que a modulação da via IL-4R/STAT6 e da população de células Th17 funcione como mecanismo de evasão parasitária em HIV/LM. Estudos futuros são necessários para confirmar estes resultados.

Immunophenotypic characterisation of peripheral T lymphocytes in pulmonary tuberculosis.

BACKGROUND: The cellular immune response plays an important role in determining the outcome of infection and disease in Mycobacterium tuberculosis. Many studies of these disease interactions yield contradictory results. AIM: This study aims at determining the changes that take place in the subpopulations of T lymphocytes in the blood of patients with pulmonary tuberculosis (TB). SETTINGS AND DESIGN: This cross-sectional study was done at King Khalid University Hospital, Riyadh, Saudi Arabia. MATERIALS AND METHODS: Flow cytometry was used to determine the absolute numbers and percentages of T CD3, T CD4, T CD8, T CD19 and natural killer (NK) T cells in 54 patients with active pulmonary TB before the commencement of treatment and in 25 healthy PPD negative volunteers. STATISTICAL ANALYSIS: Statistical Package for Social Sciences (version 11.5) was used for analysis. RESULTS: There were significant differences in the values of CD3, CD4 and NK T cells among the groups. The numbers of CD3 and CD4 cells were lower in subjects than in controls [1091.9 +/- 321.4 vs. 1364.6 +/- 251.2; P < 0.001 and 639.8 +/- 285 vs. 822 +/- 189.9; P < 0.004, respectively] while numbers of NK T cells were much higher in patients than in controls (410.7 +/- 286 vs. 182.3 +/- 140; P < 0.001). The numbers of CD8 cells were not significantly changed with disease (609 +/- 233.5 in subjects and 613.4 +/- 170.3 in controls P = 0.761). CONCLUSION: There are significant changes in the cellular immune response particularly affecting the CD3, CD4 and NK T cells with the development of pulmonary TB. Therefore, further studies of these changes may have important implications on the development of diagnostic tools, vaccines and treatment modalities.

T-lymphocyte subpopulations in tuberculosis.

BACKGROUND: Tuberculosis is associated with both qualitative and quantitative defects in the cell mediated immune response. The changes that occur in the lymphocyte profile in blood in children with tuberculosis are not well understood. DESIGN: Prospective study. SETTING: Referral hospitals. METHODS: Lymphocyte subpopulations were determined by flow cytometry in 17 healthy tuberculin positive children, in 22 children with newly diagnosed pulmonary tuberculosis and in 8 of these children after antituberculosis therapy. RESULTS: Absolute numbers and percentages of CD3+ and CD4+ T cells were reduced in children with tuberculosis, compared to controls. CD4+ counts increased significantly following antituberculosis treatment, compared to baseline values. In contrast, the proportion of T cells expressing the gdT cell receptor was similar in tuberculosis patients and controls. CONCLUSION: Children with tuberculosis have a systemic decrease in the proportion and number of CD3+ and CD4+ T cells which reverses during therapy.

Phenotypic and functional analysis of bovine gammadelta lymphocytes

The studies have provided the first comprehensive comparison of the factors regulating activation and proliferation of WC1+ and WC1- gammadelta T cells. The investigation has shown that accessory molecules essential for activation and function of WC1+ and WC1- gammadelta T cells and the sources and roles of cytokines in activation of gammadelta T cells through the T cell receptor (TCR). The study has also shown that the role of cytokines in activation and function of gammadelta T cells activated indirectly through cytokines secreted by ab T cells, accessory cells and antigen presenting cells (APC). Cytokines were differentially produced by subpopulations of gammadelta T cells under different conditions of activation. The investigation obtained in this study has revealed that factors account for activation and proliferation of gammadelta T cells in cultures designed to study MHC-restricted responses to antigens. Evidence obtained here has shown there is biological relevance to activation under these culture conditions that points to potential regulatory and effector functions of gammadelta T cells. The investigations have also provided the information needed to begin identifying and characterizing antigens recognized by the TCR repertoires of WC1+ and WC1- gammadelta T cells. Finally, the investigations have provided the information needed to begin analysis of the mechanisms by which gammadelta T cells modulate MHC restricted immune responses to pathogens and derived vaccines.

Estudio de las subpoblaciones linfocitarias y función cooperadora de linfocitos T en pacientes con lupus eritematoso sistémico / Studies of the lymphocyte populations and T helper function in patients with systemic lupus erythematosus

El Lupus Eritematoso Sistémico (LES) se caracteriza por la producción de una serie de autoanticuerpos y diferentes alteraciones de la inmunidad celular. Se estudiaron 10 pacientes con LES recién diagnosticados y sin tratamiento (LES-1) y 7 de ellos aproximadamente 6 meses después, clínicamente inactivos con bajas dosis de Prednisona (LES-2) Se realizó evaluación clínica, determinación de poblaciones linfocitarias y función cooperadora de linfocitos T. El grupo LES-1 mostró una reducción significativa de las poblaciones linfocitarias y aumento de la función cooperadora de linfocitos T al compararlos con los controles, el grupo LES-2 presentó disminución no significativa de los mismos parámetros al compararlos con LES-1. Se concluye: 1. No existe mayor diferencia en los parámetros inmunológicos estudiados con enfermedad clínicamente inactiva, 2. Las diferencias fundamentales existen entre LES-1 y el grupo control

Lymphocyte subset T4/T8 ratio in systemic lupus erythematosus: correlation with disease activity, laboratory abnormalities and treatment.

Lymphocyte subsets were studied in forty-nine patients with SLE using monoclonal antibodies and flow cytometry. A decrease in T4+ reactive cells (helper/inducer) was the most frequent observation. Decreased T4/T8 ratios were seen in patients with increasing clinically active disease, patients with positive anti-DS-DNA, positive anti-RNP antibodies and patients with low CH50 activity. However, low T4/T8 ratios were seen in patients with negative anti-Sm. Low T4/T8 ratios were also observed in patients taking prednisone at more than 10 mg/day and in patients treated with immunosuppressive drugs.

Studies on Lymphocyte Subpopulations and Cell-mediated lmmunity in Patients with Chronic Renal Failure

Several parameters of cell-mediated immunity thirty-eight patients with end stage chronic renal failure were measured including total lymphyocytes, B-and T-lymphocytes, T-cell subsets and the mitogenic reponse to PHA and Con A at three different times; before dialysis, 3 months and 12 months after dialysis treatment. There were no significant differences in the absolute numbers of peripheral leukocytes between each patient and the control group. But the absolute numbers of lymphocytes of each patient group were significantly reduced compared to the control group (p< 0.01). The proportion of peripheral blood active T cells and helper T cells was significantly reduced both in the predialysis uremic and dialysis populations compared to the control group, although the helper/suppressor(OKT4/OKT8) ratio was not different between each patient and the control group except for a lower ratio in the hemodialysis 12 month follow-up group (HD 12M). With respect m the PHA and Con A stimulation tests, the stimulation indices of the predialysis and hemodialysis groups were significantly lower than those of the control group. However, patients on continuous ambulatory peritoneal dialysis (CAPD) exhibited a normal mitogenic response and a lower suppressor cell removal index compared to the patients on hemodialysis, suggesting an improved cell-mediated immunity in the patients undergoing CAPD.

Immunological changes and recovery in patients with dengue haemorrhagic fever.

In studying the immunological changes in dengue haemorrhagic fever, three phases of investigations had been carried out. During the earlier phase of investigation, significant immunological findings were obtained, namely the elevation of immunoglobulins, activation of complements, formation of circulating-immune-complexes and diminished number of T lymphocytes. The changes tended to recover during the convalescent phase. During the second phase of investigation, the extended studies revealed further confirmation of T cell impairment during the acute phase which tended to recover during the convalescent phase. Elevated number of Fc-receptor- and C3-receptor-bearing cells was also observed in some patients, variedly occurred during the acute or the convalescent phase. Elevated number of B cells was only found in small proportion. Significantly high number of activated RNA-rich lymphocytes was found in almost the half of patients. The virus-lymphocyte interaction has been demonstrated by the detection of viral antigen on the surface of lymphocytes in a proportion of patients. The circulating-immune-complexes was shown to contain viral (DEN-1) antigen. During the third phase of investigation, the impairment of T cells was further analyzed on their regulatory T populations. Impairment of total T lymphocytes, helper-T and suppressor-T was detected during the acute phase and tended to recover during the convalescent phase. The reversed changes occurred on B cells, The immunological changes and recovery are considered to be related to the stimulatory and suppressive effects of the dengue virus and regulatory mechanism.

Subpoblaciones de linfocitos T determinadas mediante anticuerpos (AC) monoclonales en población infantil: variable dependiente de la edad / T-lymphocyte subpopulations determined by monoclonal antibodies in children: range dependent of age

Se determinó, mediante el uso de anticuerpos monoclonales, el rango normal de subpoblaciones de linfocitos T, en sangre periférica de una población infantil chilena (1 mes a 13 años). Los resultados obtenidos en el estudio de 25 niños sanos demuestran que el porcentaje de células OKT4 fue más elevado en niños entre 1 y 24 meses de edad, y el OKT8 significativamente inferior al comparar con niños de mayor edad (61 a 157 meses). La relación de células OKT4/OKT8 disminuye al aumentar la edad alcanzando valores cercanos al adulto alrededor de los 5 años de vida. Estos resultados sugieren que la interpretación de las subpoblaciones de LT en niños debe tomar en consideración la edad

Alteraciones inmunológicas en pacientes con púrpura trombocitopenica idiopática crónica / Immunological changes in patients with chronic idiopathic thrombocytopenic purpura

En este trabajo se valoró la inmunidad mediada por células y los niveles de inmunocomplejos circulantes en 30 pacientes con púrpura trombocitopénica idiopática crónica (PTI). Se hallaron diversas alteraciones inmunológicas: los pacientes con PTI tenían valores relativos de linfocitos B aumentados y disminuidos los de linfocitos T. La transformación blástica inducida por PHA estaba disminuida al igual que la función supresora T-T inespecífica inducida por Con A. Se rencontró una correlación directa entre los recuentos plaquetarios y la función supresora (r=0,61, p<0,01). El nivel de IgG asociada a la membrana plaquetaria era más alto en aquellos pacientes con marcada depresión de la actividad supresora. Estos resultados no estaban relacionados a la edad y sexo de los pacientes ni al tratamiento o evaluación de la enfermedad

Regulación de la respuesta inmune mediada por interacciones celulares: su relación con la inmunidad anti-tumoral / Regulation of the immune responce mediated by cellular interactions: its relation to anti-tumor immunity

Se describen los posibles mecanismos de regulación de la respuesta inmune en el ratón, fundamentalmente los mediados por interacciones celulares. Los macrófagos presentan el antígeno a células T que se caracterizan por poseer el marcador de superficie Lyt 1, y reconocen a antígenos del complejo mayor de histocompatibilidad de clase II en al superficie de la célula presentadora del antígeno. Estos macrófagos liberan un factor solubre, 1a IL-1, que activa a las células Lyt 1 las que como consecuencia de ello liberan otro factor soluble, 1a IL-2, que permite la proliferación y diferenciación celular. Las células T participan en la regulación de la respuesta inmune. Se sugiere que las células T colaboradoras estimulan una cascada de células T supresoras, cada una de ellas activando la próxima hasta llegar a la última que transmite una señal que reduce la actividad de las células colaboradoras. Por otra parte, las células supresoras hiperactivadas pueden reestimular a las células colaboradoras y así, por un mecanismo de feed-back se produciría la reactivación de las células B productoras de anticuerpos, de las células citotóxicas o de los macrófagos. También los macrófagos son esenciales en la regulación de la respuesta inmune ya que factores supresiones de las células que los producen a las células aceptoras. El sistema regulatorio también actuaría contra los antígenos específicos de tumor, los que pueden estar constituídos por antígenos que no se expresen al mismo tiempo, en la misma cantidad o en la misma localización que en las células normales

Tr, T mu and B lymphocytes in erythema nodosum leprosum reactions of leprosy.

Enumeration of sub-population of T cells with receptors for Fc portion of Ig G (Tr) and Fc portion of Ig M (Tu) and B lymphocytes in the peripheral blood of 39 Lepromatous, 44 ENL and 22 Post ENL patients were undertaken. ENL patients showed significant decreased Tr cell percentage than Lepromatous and Post ENL patients. Although T mu cell percentage was lowered in ENL patients, a relatively elevated T mu/Tr ratio was found than Lepromatous and Post ENL patients indicating elevation of helper activity in ENL. B cells did not register any change in the three stages.

Subpoblaciones de linfocitos T en individuos con riesgo de contraer síndrome de inmunodeficiencia adquirida / Subpopulations of T-lymphocytes in patients with risk of develop acquired immunodeficiency syndrome

Mediante el uso de anticuerpos monoclonales, se estudiaron las subpoplaciones de linfocitos T en 15 sujetos, en su mayoría varones homosexuales sanos, comparándolos con un grupo control. Se analizaron individualmente y en cifras absolutas los valores de linfocitos B, T, T ayudadores (T4), T supresores/citotóxicos (T8) y la relación T4/T8, para cada grupo. Los valores del grupo control son equiparables a los publicados por autores extranjeros. En el grupo de estudio se encontró que en más de la mitad de los individuos existían alteraciones en alguna de las subpoblaciones de linfocitos, expecialmente el descenso de los linfocitos T4 y en pocos, un incremento de los linfocitos T8

Caracterizaçäo das subpopulaçöes de linfócitos T definidos por anticorpos monoclonais na cardiopatia chagásica / Characterization of T-lymphocytes subpopulations determinate by monoclonal antibodies in chronic Chagas-cardiopathy

Com a finalidade de avaliar o perfil de linfócitos T (OKT3+) e de suas subpopulaçöes auxiliar-indutora (OK4+) e citotóxico-supressora (OKT8+), foram estudados 25 pacientes portadores de cardiopatia chagásica crônica, sendo 16 do sexo feminino e 9 do masculino, com idades variando de 27 a 65 anos (x = 46,9) e comparados com igual número de indivíduos normais com idades de 23 a 68 anos (x = 46,3). A caracterizaçäo dos linfócitos foi feita por tratamento da fraçäo mononuclear do sangue periférico com anticorpos monoclonais específicos para cada tipo celular e contagem das células marcadas por imunofluorescência indireta. Os valores obtidos no grupo controle foram: OKT3+ = 56,0 + ou - 7,6; OKT4+ = 36,3 + ou - 5,6; OKT8+ = 18,2 + ou - 4,3 e relaçäo T4/T8 = 1,97 + ou - 0,25. Os valores do grupo com cardiopatia chagásica crônica foram OKT3+ = 53,3 + ou - 10,6; OKT4+ = 36,2 + ou - 10,9; OKT8+ = 17,7 + ou - 6,5 e relaçäo T4/T8 = 2,04 + ou - 0,95. Estatisticamente, näo foram observadas diferenças entre as duas populaçöes. Porém, quando a populaçäo chagásica foi analisada isoladamente, notou-se diferença significativa entre os pacientes do sexo masculino e feminino, com valores de OKT4+ de 29,3 + ou - 6,4 e 40,1 + ou - 11,0 (p < 0,05); valores de OKT8+ de 21,1 + ou - 4,7 e 16,5 + ou - 6,0 (p < 0,02) e relaçöes T4/T8 de 1,33 + ou - 0,33 e 2,57 + ou - 1,0 (p < 0,002), respectivamente. Embora estes dados sugiram uma relaçäo entre células T imunorreguladoras e cardiopatia chagásica crônica, nenhuma correlaçäo foi encontrada entre a alteraçäo das subpopulaçöes de células T e os dados clínicos dos pacientes estudados