Relationships between phenotype and function of blood cd4+ t-cells and ascending thoracic aortic aneurysm: an experimental study

Abstract Introduction: Non-familial ascending thoracic aorta dilation and aneurysms (TAAs) are silent diseases in elderly patients. Histopathology revealed that functionally polarized infiltrating CD4+ T-cells play a key role in aortic wall weakening. Objective: To evaluate the possible associations between phenotype and cytokine production of circulating CD4+ T-lymphocytes and the presence of TAA in patients with aortic valve disease (AVD). Methods: We studied blood samples from 10 patients with TAA and 10 patients with AVD. Flow cytometry was used to quantify: a) CD4+ T-lymphocytes surface expression of CD25, CD28, and chemokine receptors (CCR5, CXCR3, CX3CR1); b) fractions of in vitro stimulated CD4+ T-cells producing cytokines (interferon gamma [IFN-γ], interleukin [IL]-17A, IL-21, IL-10); c) CD4+CD25highFoxP3+ regulatory T-cells (Treg) fraction. Enzyme-linked immunosorbent assays (ELISA) were performed for cytokines (IFN-γ, IL-6, IL-10, IL-17A, IL-23, transforming growth factor beta [TGF-β]) and chemokines (RANTES, CX3CL1). Results: The total CD4+CD28±CD4+/CX3CR1+ T-cells fraction was higher (P=0.0323) in AVD (20.452±4.673) than in TAA patients (8.633±2.030). The frequency ratio of CD4+ T-lymphocytes producing IFN-γ vs. IL-17A+IL-21 cytokine-producing CD4+ T-cells was higher (P=0.0239) in AVD (2.102±0.272) than in TAA (1.365±0.123) patients. The sum of CD4+CD28±CD4+/CX3CR1+ T-cells correlated positively with values of the previous cytokine ratio (P=0.0002, R=0.732). The ratio of CD4+CD28±CD4+/CX3CR1+ T-cells vs. Treg was higher (P=0.0008) in AVD (20.859±3.393) than in TAA (6.367±1.277) patients. Conclusion: Our results show that the presence of TAA in subjects with AVD is associated with imbalance between phenotypic and cytokine-producing subsets of circulating CD4+ T-lymphocytes, prevalently oriented towards a pro-fibrotic and IFN-γ counteracting effect to functional polarization.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 + or - 2.7 per cent) as compared with lesions undergoing spontaneous healing (mean = 17.8 + or - 2.2 per cent). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 + or - 3.8 and 15.3 + or - 3.0 per cent, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Apoptosis e infección por el virus de la inmunodeficiencia humana (HIV) / [Apoptosis and human immunodeficiency virus infection (HIV]

Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.

Presentación de cinco casos de linfocitopenia CD4+ idiopática / Report of five cases of idiopathic CD4+ lymphocytopenia

Introducción: el objetivo de este trabajo es llamar la atención sobre una inmunodeficiencia recientemente descripta y ofrecer una propuesta de estudio y tratamiento. Material y métodos: período de estudio: enero de 1998 a agosto de 2000. Presentamos 5 pacientes de entre 23 y 86 años de edad (media 51,2 años), todos de sexo femenino, que fueron estudiadas por presentar infecciones a repetición (micóticas, virales y bacterianas). Se les realizó determinación de VIH por ELISA, pruebas cutáneas para antígenos habitualmente reconocidos por el organismo, medición de linfocitos CD4+ y CD8+ por partículas magnéticas. Proteinograma electroforético, dosaje de inmunoglobulinas plasmáticas por inmunodifusión radial. Se efectuó tratamiento con timomodulina 60 mg por día. Conclusiones: al momento del diagnóstico se observó: pruebas cutáneas hipoérgicas, VIH negativo y disminución de linfocitos CD4+. Posterior al tratamiento con timomodulina, se observó buena respuesta clínica, mejoría de las pruebas cutáneas y elevación de los linfocitos CD4+ en todas las pacientes

Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

Infección por virus de la inmunodeficiencia humana: aumento de eosinófilos en sangre periférica durante la progresión de la infección / Human immunodeficiency virus infection: increse of eosinophils in peripheralblood during progress of the infection

En este estudio se evaluó el nivel de eosinófilos en sangre periférica de individuos HIV-seropositivos y su relación con el estado inmune y las manifestaciones clínicas. Se incluyeron 79 pacientes agrupados según el nivel absoluto de linfocitos (L) CD4+ y manifestaciones clínicas asociadas en; 1) 23 pacientes con infección asintomática (IA) y > 500 L.CD4+/mm3, 2) 24 pacientes con < 200 L.CD4+/mm3 y manifestaciones clínicas asociadas (SIDA) y 3) 32 pacientes con IA y > 500 L.CD4+/mm3 que durante el estudio evolucionaron a SIDA. Los pacientes fueron evaluados durante un período medio de 27 meses. Se consideró aumento de eosinófilos a cifras porcentuales ò 5 por ciento que persistieron en tres o más estudios consecutivos, debido a que la leucopenia presente en pacientes con SIDA no permitió evidenciar el aumento absoluto de eosinófilos. En pacientes con SIDA (Grupo 2) hubo un aumento significativo del porcentaje de eosinófilos respecto de pacientes con IA (Grupo 1) (6,0 ñ 0,7 por ciento vs. 3,3 ñ 0,2 por ciento, p < 0,05). La incidencia de cifras porcentuales ò 5 por ciento de eosinófilos fue de 37,5 por ciento en pacientes con SIDA y 21,7 por ciento en pacientes con IA. En estos pacientes, las cifras porcentuales fueron de 10,0 ñ 1,2 por ciento y 6,3 ñ 0,6 por ciento respectivamente. En pacientes con IA y que durante el estudio evolucionaron a SIDA (Grupo 3) hubo un comportamiento similar en los niveles de eosinófilos como el observado en pacientes con IA y pacientes con SIDA. En pacientes con IA y aumento de eosinófilos hubo una disminución significativa (p < 0,05) de inmunoglobulinas A y E séricas respecto de pacientes sin aumento de eosinófilos. El aumento de eosinófilos no estuvo relacionado con las manifestaciones clínicas y tratamientos. En conclusión, estos hallazgos indican que el aumento de eosinófilos durante la evolución de la infección por HIV tiene un significado variado y puede ser considerado en el contexto del estado de inmunodeficiencia y las manifestaciones clínicas asociadas

Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41

The calcium inophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4-CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibitis the increased intracellular calcium promoted by ionomycin but not the increase promoted by antiCD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4-CD8- cells, and this is due to cytosolic calcium mobilization

Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC) and cloned. These T cell clones (TCC) were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86 por cento). On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83 por cento), bradycardia with megacolon (75 por cento) and bradycardia (75 por cento). Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

Diarrea crónica y Cryptosporidium en pacientes diabéticos con subpoblación linfocitaria normal: informe de dos casos / Chronic diarrhea and Cryptosporidium in diabetic patients with normal lymphocyte subset counts: report of two cases

Se describen dos casos de pacientes diabéticos con cuentas normales de linfocitos TCD4+, que presentaban diarrea crónica y en los cuales se detectó Cryptosporidium en heces. En ambos casos las pruebas serológicas para VIH resultaron negativas. El hecho de que estos pacientes desarrollaran una patología que se observa comunmente en presencia de cuentas bajas de linfocitos TCD4+, sugiere que una alteración inmunológica distinta de la celular pudiera estar involucrada en la patogénesis de esta infección. Los autores sugieren que la búsqueda intencionada de Cryptosporidium debe considerarse en el estudio de la diarrea crónica del paciente diabético

Enhanced phagocyte chemiluminescence in asymptomatic HIV infection.

Seventeen asymptomatic HIV infected patients were studied for their phagocyte function in vitro, in comparison with that of eight normal healthy persons. Chemiluminescence was measured using whole blood by means of a microtitreplate luminometer. Light indices, cumulative light indices and rapidity of responses were recorded. The patients had a lower phagocyte count (13.17 +/- 0.85 x 10(9)/l) but a more rapid and intense chemiluminescence response. The latter was demonstrated by a higher peak light index and cumulative response. The observed enhanced phagocyte activity may reflect an early failure of T cell regulatory functions, or a compensatory mechanism in response to the underlying immunodeficiency.