José Peña

NK phenotypic markers and IL2 response in NK cells from elderly people

Immunosenescence is a process that primarily affects the T cell compartment of the immune system, although age-associated immunological alterations have also been demonstrated in the NK cell phenotype and function. A significant expansion in the number of NK cells is found in aging. The NK cytotoxic capacity of total peripheral blood lymphocytes is also well preserved, not only in healthy elderly people but also in centenarians. However, NK cell killing of K562 is impaired when considered in a per-cell basis, and this defect is associated with defective signal transduction after activation more than a diminished conjugate formation or killing capacity. We have studied the phenotype of NK cells in elderly donors fulfilling the Senieur criteria. We have also studied the capacity of these cells to be activated by IL2 when different NK cell functions, other than cytotoxicity, are considered. Our results confirm the increased percentage of NK cells in the elderly due to the expansion of the CD56dim subset that also show an altered pattern of activation markers, whereas no differences were found in the CD56bright subset. The response of NK cells to IL2 was found to be impaired when proliferation, expression of CD69, and Ca2+ mobilization were considered, whereas TNF-alpha production was not significantly affected. These results suggest that human NK cells do not escape the aging process, although senescence have a differential effect on distinct NK cell biological functions, ranging from severe to negligible impairment, depending on the parameters considered.

CD69 is a stimulatory receptor for natural killer cell and its cytotoxic effect is blocked by CD94 inhibitory receptor

CD69 is a differentiation antigen expressed shortly after activation on T lymphocytes and other cells of haematopoietic origin, including natural killer (NK) cells. The function of CD69 on T lymphocytes acting as a costimulatory molecule in proliferation and lymphokine secretion is well established. NK cells express CD69 after activation by different stimuli such as phorbol 12‐myristate 13‐acetate (PMA), interleukin (IL)‐2, IL‐12, interferon‐α (IFN‐α) or anti‐CD16 monoclonal antibodies (mAbs). However, although it has been shown that CD69 triggers NK‐cell‐mediated cytolytic activity, its effect on other NK‐cell functions has not been studied. Furthermore, the possible interaction of CD69 triggering with other C‐lectin type inhibitory receptors is not known. Thus, the objective of this work is to determine whether CD69‐mediated NK cytotoxicity can be regulated by CD94 inhibitory receptor and the role of CD69 on other NK‐cell functions different of cytotoxicity. The results show that CD69‐mediated NK cytotoxicity can be abrogated by CD94 stimulation in NK cells expressing the CD94 inhibitory form of the receptor, indicating that CD94 regulates the cytotoxic events initiated by a wide variety of NK activatory receptors. We also show that anti‐CD69 mAbs, not only triggered NK cytotoxicity, but also induce NK‐cell proliferation, CD25 and intracellular adhesion molecule‐1 (ICAM‐1) expression, TNF‐α production and Ca2+ mobilization in preactivated NK cells. These results suggest that CD69 plays a crucial role in NK‐cell function contributing to sustain NK‐cell activation, as it has been previously demonstrated in T cells.

Selective depletion of CD56dim NK cell subsets and maintenance of CD56bright NK cells in treatment-naive HIV-1-seropositive individuals

Human immunodeficiency virus-1 (HIV-1) infected patients show a gradual loss of natural killer (NK) cells that correlates with disease progression. However, the effect of HIV-1 infection on different NK cell subsets has not been fully characterized. In healthy individuals most NK cells are CD3−CD56+ and two different subpopulations, CD56dim and CD56bright, can be distinguished by the mean fluorescence intensity. Although it was originally suggested that CD56bright NK cells represent the precursors of the CD56dim subpopulation, recent cumulative data indicate that CD56bright and CD56dim NK cells are phenotypically, functionally, and developmentally different NK cell subsets. In this study, the analysis of CD56bright and CD56dim NK subsets showed that neither the number nor the phenotype of CD56bright NK cells were significantly altered in treatment-naive HIV-1-infected individuals, whereas the number of CD56dim NK cells was decreased. We also have studied NK cell subsets defined by the expression of CD56 in combination with CD16, CD161, or CD94 molecules. Our results demonstrated a preferential decrease of CD3−CD56+ NK cells coexpressing CD16 and CD161 but lacking CD94 molecules. On the contrary an increased percentage of NK cells that do not express CD56 molecules but express CD16, CD161, or CD94 was also found in HIV-1-infected individuals. As it has been proposed that these CD56-negative NK cells expressing other NK cell receptors represent immature NK cells with low cytolytic capacity, our results support that a defective differentiation from immature CD56 negative NK cells to mature CD56dim NK cells occurs in HIV-1 infection.

Natural killer cells in healthy aging.

Immunosenescence is a process that affects all cell compartments of the immune system. Age-associated changes have been demonstrated not only on T lymphocytes but also in different aspects of the innate immunity including natural killer (NK) cells. A significant expansion in the percentage of NK cells showing a mature phenotype has been found in healthy elderly donors, and the NK-cytotoxic capacity of total peripheral blood lymphocytes is well preserved in these individuals. However, NK-cell killing of K562 is impaired when considered on a per-cell basis. Furthermore, NK cells from elderly people show a decreased proliferative response to interleukin 2 and a parallel impaired expression of the CD69 activation antigen. The response to interleukin 2 of NK cells from aged donors is also impaired in terms of their capacity to kill NK-resistant cell lines, but not when K562 killing, perforin synthesis, or tumor necrosis factor alpha production are considered. Therefore phenotypic and functional alterations can be shown in NK cells in healthy aging. These changes are compatible with the expansion of a mature NK subset.

Impact of protease inhibitor therapy on HIV-related oropharyngeal candidiasis.

ObjectiveTo determine the relationship between antiretroviral therapy and changes in prevalence and amount of oropharyngeal candidiasis (OPC) and skin test reactivity for delayed type hypersensitivity. DesignObservational cohort. SettingUniversity-based public hospital AIDS clinic. PatientsAdults with advanced HIV infection who had been taking nucleoside transcriptase inhibitor drugs but had not taken a protease inhibitor and who started antiretroviral treatment with ritonavir. Main outcome measuresOPC lesions score, oral candidal colonization, oral candidal quantification, skin test reactivity for delayed type hypersensitivity (purified protein derivative, candidal and streptokinase antigens), plasma HIV RNA and CD4 cell count at weeks 8, 16 and 48 weeks. ResultsIn the 99 patients who entered the study, there was a significant reduction in the HIV plasma RNA (mean log decrease from baseline at 48 weeks 0.88) and a significant increase in CD4 cell counts (mean CD4 cell increase from baseline at 48 weeks 128 × 106 cells/l). Only 17% of patients had < 200 copies/ml HIV RNA at 48 weeks. There were significant decreases in the prevalence of OPC lesions (31% at baseline to 1% at 48 weeks;P  < 0.001), and in oral candidal loads [2226 to 811 colony-forming units (CFU)/ml;P  = 0.0171]. The percentage of patients with at least one positive skin test increased significantly (6 to 28%;P  < 0.05). Patients whose CD4 lymphocyte count was > 200 × 106 cells/l at 48 weeks had significantly lower oral candidal loads and were more likely to have a positive skin test than patients whose CD4 cell count was < 200 × 106 cells/l. ConclusionIn patients with advanced HIV infection, antiretroviral treatment including a protease inhibitor has a positive impact in the natural history of OPC. This positive impact appears to be correlated with a better immunological function and occurs despite continuous HIV replication.

Vα24+ NKT cells are decreased in elderly humans

Natural killer T (NKT) cells represent a novel cell lineage characterized by the restricted expression of an invariant TCRα chain encoded by Vα24/JαQ gene segments in humans and Vα14/Jα281+ in mice. Different aspects of the immune response are severely affected by age. Thus, we have studied the effect of aging on NKT cells from healthy elderly individuals. Our results demonstrated a decreased percentage of CD3+Vα24+ cells in peripheral blood from elderly donors, whereas mainstream T lymphocytes showed an age-associated decrease in the expression of CD28, the vast majority of CD3+Vα24+ cells from old individuals were CD28+. A significant increase in the percentage of Vα24+ cells with the CD4−CD8+ phenotype was also found in the elderly, indicating a redistribution of Vα24+ subsets according to the CD4/CD8 phenotype. Given the important immunoregulatory role of these cells, the decrease of NKT cells will contribute to the deleterious immune response in the elderly.

Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals.

Dysfunction of cytotoxic activity of T and natural killer (NK) lymphocytes is a main immunological feature in patients with AIDS, but its basis are not well understood. It has been recently described that T and NK cell‐mediated cytotoxicity can be regulated by HLA killer inhibitory receptors (KIR). In this work, we have determined on cytotoxic T cells and NK cells from HIV‐1‐infected individuals the expression of the following KIR molecules: p58, p70, and ILT2 (immunoglobulin‐like family KIR) as well as CD94 and NKG2A (C‐lectin‐type family KIR). With some exceptions, no significant changes were found on the expression of immunoglobulin‐like KIR in either CD8+ or CD56+ cells. Interestingly, the percentages of CD8+ and CD56+ cells expressing CD94 were significantly increased in these individuals. We also show that, in vitro, IL‐10 up‐regulates CD94 expression on CD8+ and CD56+ cells obtained from normal individuals, suggesting that the augmented expression observed in HIV‐infected individuals could be related to the high levels of IL‐10 previously described in HIV‐1‐infected individuals.

CD8 T cells expressing NK associated receptors are increased in melanoma patients and display an effector phenotype

CD8+ T cells can express NK-associated receptors (NKRs) that may regulate their cytolytic function. We have characterized the expression of several NKRs on peripheral blood CD8+ T cells from melanoma patients and compared them to age-matched healthy donors. The analysis performed includes HLA class I specific receptors (KIRs, LILRB1 and CD94/NKG2) and other NK receptors like CD57, CD56 and CD16. Melanoma patients showed a higher variability in the expression of NKRs on circulating CD8+ T cells than age-matched healthy donors. NKR expression on CD8+ T cells from melanoma patients showed a significant increase of KIR2DL2/L3/S2 (mAb gl183), CD244, CD57, CD56 and CD16. We have also found an increase of CD8+ CD28− CD27− T cells in melanoma patients. This subset represents terminally differentiated effector cells expressing CD244 and high levels of perforin. The expression of NKRs was also mainly restricted to this T cell subset. Altogether, circulating CD8+ T cells from melanoma patients display a distinct phenotype characterized by downregulation of costimulatory molecules and higher expression of NKRs. We suggest that the increased expression of NKRs on T cells may contribute to the final outcome of the immune response against melanoma both stimulating or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptor function and enhancing activating receptors may represent new strategies with therapeutic potential against melanoma.

Transcranial magnetic stimulation attenuates cell loss and oxidative damage in the striatum induced in the 3‐nitropropionic model of Huntington's disease

An investigation was conducted on the effect of transcranial magnetic field stimulation (TMS) on the free radical production and neuronal cell loss produced by 3‐nitropropionic acid in rats. The effects of 3‐nitropropionic acid were evaluated by examining the following changes in: the quantity of hydroperoxides and total radical‐trapping antioxidant potential (TRAP), lipid peroxidation products, protein carbonyl groups, reduced glutathione (GSH) content, glutathione peroxidase (GSH‐Px), catalase and succinate dehydrogenase (SDH) activities; total nitrite and cell death [morphological changes, quantification of neuronal loss and lactate dehydrogenase (LDH) levels]. Our results reveal that 3‐nitropropionic acid induces oxidative and nitrosative stress in the striatum, prompts cell loss and also shows that TMS prevents the harmful effects induced by the acid. In conclusion, the results show the ability of TMS to modify neuronal response to 3‐nitropropionic acid.

Time course and prognostic significance of hemostatic changes in sepsis: relation to tumor necrosis factor-alpha.

OBJECTIVES To describe the time course and prognostic significance of tumor necrosis factor-alpha (TNF-alpha) levels and hemostatic abnormalities in clinical sepsis. DESIGN Prospective, observational study with sequential measurements in an inception cohort. SETTING An emergency department in a university teaching hospital. Patients were followed up until they either left the hospital or died. PATIENTS During a 1-yr period, 43 adult patients were selected from all emergency department patients who met the established criteria for sepsis. Excluded were patients with either organ dysfunction or septic shock at the time of admission. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Blood samples were collected serially (day of admission and on days 3, 5, and 7) to determine TNF-alpha, platelet count, fibrinogen, factor VII, antithrombin III, tissue-type plasminogen activator activity, plasminogen activator inhibitor activity, plasminogen, and alpha2-antiplasmin. Fibrinopeptide A was measured only on the day of admission. Data were analyzed to determine whether admission values or serially obtained values within 7 days were useful in predicting outcome. Thirteen patients died and 30 survived. On admission, assay values indicated that platelet count and antithrombin III were significantly lower than normal (as observed in 50 healthy adults). Fibrinogen, plasminogen activator inhibitor type 1, tissue-type plasminogen activator, fibrinopeptide A, and TNF-alpha were higher than normal, whereas concentrations of factor VII, plasminogen, and alpha2-antiplasmin were in the normal range. No differences were detected in the admission values between survivors and nonsurvivors, except for antithrombin III. However, subsequent values of some variables demonstrated a difference between survivors and nonsurvivors. Survivors showed increasing platelet count and antithrombin III values compared with nonsurvivors, in whom the values remained low, with no significant changes during the study period. High TNF-alpha levels were found in both groups, but only survivors experienced progressive decrease during the observation period. CONCLUSIONS Early clinical sepsis is characterized by high plasma levels of TNF-alpha and by activation of the coagulation and fibrinolysis systems. Longitudinal analysis of some variables (antithrombin III, platelet count, and TNF-ea) showed some differences with time between the survivor and nonsurvivor groups, but we feel that such differences were not large enough to be predictive in individual patients.