Detection of Apoptosis in Peripheral Blood Cells of 31 Subjects Affected by Down Syndrome Before and After Zinc Therapy

Thirty-one patients affected by Down syndrome (DS) were investigated to study the presence of apoptosis in peripheral blood cells in relation to the plasma levels of zinc. Twelve patients had undergone therapy with ZnSO₄, while the remaining 19 were untreated. The presence of programmed cell death was evaluated by means of electron microscopy, in situ nick translation (NT), and agarose gel electrophoresis of DNA. These approaches evidenced the presence of apoptosis in peripheral blood cells of patients before therapy with ZnSO₄, while after zinc supplementation there was a reduction in the number of apoptotic cells. These results suggest that the process of programmed cell death in peripheral blood cells of patients with Down syndrome is related to the plasma levels of zinc ion.     

Programmed Cell Death of Peripheral Myeloid Precursor Cells in Down Patients: Effect of Zinc Therapy

Hemopoietic stem cell differentiation represents the primary rule of self-renewal, proliferation, and specialization modulated by several mechanisms, including growth factors, cell interactions, and bioavailability of various ions, especially Ca²⁺ and Zn²⁺. Apoptotic death, during normal cell turnover, has been widely studied and is recognized as an important pathway for clonal deletion in the hemopoietic system. Multi-parametric analyses have shown that subjects with Down syndrome show low levels of plasmic zinc associated with the presence of immature myeloid cells in the peripheral blood. This arrangement is repaired by in vivo zinc therapy. This study presents morphological and biochemical analyses to show that ZnSO₄ therapy induces the disappearance of peripheral myeloid precursor cells by a programmed cell death mechanism. The programmed zinc-therapy-induced cell death presumably provides a simple way to regulate the myeloid differentiation selecting appropriate cells.     

Programmed Cell Death of Peripheral Myeloid Precursor Cells in Down Patients: Effect of Zinc Therapy

Hemopoietic stem cell differentiation represents the primary rule of self-renewal, proliferation, and specialization modulated by several mechanisms, including growth factors, cell interactions, and bioavailability of various ions, especially Ca²⁺ and Zn²⁺. Apoptotic death, during normal cell turnover, has been widely studied and is recognized as an important pathway for clonal deletion in the hemopoietic system. Multi-parametric analyses have shown that subjects with Down syndrome show low levels of plasmic zinc associated with the presence of immature myeloid cells in the peripheral blood. This arrangement is repaired by in vivo zinc therapy. This study presents morphological and biochemical analyses to show that ZnSO₄ therapy induces the disappearance of peripheral myeloid precursor cells by a programmed cell death mechanism. The programmed zinc-therapy-induced cell death presumably provides a simple way to regulate the myeloid differentiation selecting appropriate cells.     

HIV-1 upregulates Fas ligand expression in CD4+ T cells in vitro and in vivo: association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid.

CD4+ T-lymphocyte apoptosis has been associated with human immunodeficiency virus (HIV)-1 infection in vitro, paralleling the expression of Fas (APO-1, CD95) on peripheral blood mononuclear cells from patients with HIV disease. However, the link between Fas induction, T-cell activation, and cell death is unclear. We document, for the first time, marked upregulation of expression of mRNA for the ligand for Fas in peripheral blood mononuclear cells from HIV seropositive individuals, and demonstrate the ability of HIV infection to induce such expression in CD4+ T cells in vitro. We also define the relevance of this expression to HIV-mediated CD4+ T cell death. Our ability to downregulate Fas ligand message and suppress HIV-mediated apoptosis with aurintricarboxylic acid, a clinically used protease inhibitor with known activity against programmed cell death in other systems, may open up a new area of therapy for HIV infection.     

Spontaneous and oxidative stress-induced programmed cell death in lymphocytes from patients with ataxia telangiectasia (AT)

T cell lymphopenia in the peripheral blood lymphocytes (PBL) of patients with AT is mainly caused by a decrease of naive CD45RA+/CD4+ cells followed by a predominance of memory CD45RO+ lymphocytes. To relate these findings to the regulation of programmed cell death, we investigated the activation state and apoptotic level of PBL in 12 patients and healthy controls by flow cytometry. In accordance with previous investigations, the number of naive CD4+/CD45RA+ cells was significantly decreased in patients compared with healthy controls. This disturbed balance of CD45RA and CD45RO was also reflected in higher amounts of activated HLA-DR and CD95 expressing cells, with a concomitant decrease of Bcl-2 protected lymphocytes in the T cell population. With regard to its role in preventing oxidative-induced cell death, we analysed Bcl-2 expression and apoptosis in the presence of oxidative stress. In culture, cells of patients are more susceptible to spontaneous programmed cell death. However, in our stress-inducing system (hypoxanthine/xanthine oxidase system) the number of cells undergoing apoptosis was lower in patients' cell populations compared with controls. In addition, preliminary results suggest that Bcl-2 expression and level of spontaneous apoptosis in patients can be modified by IL-2 and interferon-gamma.     

Delayed effect of antileukemic polychemotherapy on cell death and proliferation during remission of acute lymphoblastic leukemia in children

The effect of programmed antileukemic drug therapy on cells of the tumor clone is not selective, and the drugs affect also intact bone marrow and peripheral blood cells. The cytotoxic effect of antitumor therapy is attained through triggering apoptosis and does not depend on the type of drug therapy. The treatment induces long-lasting changes in spontaneous cell death processes. The disturbances in the kinetics of cell death caused by drug therapy are compensated by changes in proliferative activity of bone marrow cells.     

Programmed cell death (apoptosis) as a mechanism of cell death in peripheral blood mononuclear cells from cats infected with feline immunodeficiency virus (FIV).

FIV is a lentivirus infection of cats which induces an immunodeficiency syndrome associated with early qualitative defects in antigen-specific T cell function and with late quantitative defects in CD4+ T lymphocytes. We have observed that peripheral blood mononuclear cells (PBMC) from FIV-infected cats have impaired survival in culture. The mechanism of this in vitro dysfunction and depletion is not known. We have proposed that inappropriate induction of programmed cell death (apoptosis) could account for these in vitro defects. Here, we report that PBMC from FIV-infected cats, with impaired T cell blastogenesis and impaired survival in vitro, undergo an active cell death upon culture, which has the morphological and biochemical characteristics of programmed cell death (PCD). Apoptosis occurred in all six asymptomatic FIV-infected cats, and in none of the nine uninfected cats, which were studied. Changes in cell morphology under both light and electron microscopy, and fragmentation of genomic DNA were characteristic for apoptosing cells. Cell death was spontaneous and occurred in the absence of any stimuli, and culture with the T cell mitogen, concanavalin A (Con A), did not significantly enhance cell death. Activation-induced cell death was inhibited, in a dose-dependent manner, by addition to the incubation medium of zinc, which has been shown to inhibit the action of endonuclease responsible for the characteristic fragmentation of DNA. Since apoptosis has recently been implicated in AIDS pathogenesis, FIV infection may prove useful to study this aspect of retroviral, in particular HIV, infection.     

Relationship between Apoptosis and Expression of Heat Shock Proteins in Peripheral Blood Lymphocytes of Patients with Myocardial Infarction

We studied activity and dynamics of apoptosis of peripheral blood lymphocytes in patients with myocardial infarction and analyzed the relationship of these processes with expression of heat shock proteins with a molecular weight of 70 kDa playing an essential role in preventing cell death. Thus, we first demonstrated activation of apoptosis in peripheral blood cells of patients with myocardial infarction compared to the control (healthy individuals) and revealed the expected negative correlation between the expression of heat shock proteins with a molecular weight of 70 kDa by lymphocytes and intensity of their death. The observed dynamics of mononuclear cell apoptosis in the peripheral blood of patients with myocardial infarction can reflect activity of programmed cardiomyocyte death in the focus of ischemic injury.     

Characteristic of readiness of thymocytes and peripheral blood lymphocytes to proliferation and apoptosis in experimental chronic liver disease

Indices of of thymocyte and peripheral blood lymphocyte readiness to apoptosis and proliferation were studied in experimental chronic liver disease of toxic and autoimmune genesis. Smears of thymocytes and peripheral blood lymphocytes were examined. Readiness to programmed cell death was determined by indirect immunofluorescence reaction with monoclonal antibodies to detect FAS/APO antigen (CD95), known to mediate apoptosis. Proliferative activity was estimated by counting nucleolar organizing regions. Indices of thymocyte readiness to apoptosis and proliferation were found to decrease in experimental animals, while similar parameters of peripheral blood lymphocytes increased. These data suggest the increased sensitivity of immunocompetent cells to activating signals, which might result in impaired immune response.     

Programmed Cell Death (Apoptosis) in Cord Blood Lymphocytes

Cord blood lymphocytes are functionally immature and have deficient immune responses. In order to determine whether the process of programmed cell death is distinct between cord blood and peripheral blood lymphocytes, we analyzed the expression of fas and bax (apoptosis promoting genes) and bcl-2 and bcl-x _L (apoptosis inhibiting genes) at protein or mRNA levels using flow cytometry and quantitative PCR methods, respectively. The susceptibility of T cell subsets from cord blood and adult peripheral blood to undergo apoptosis following restimulation with anti-CD3 or anti-Fas monoclonal antibodies was also studied. We observed that cord blood T cell subsets expressed lower levels of Fas and Bcl-2, a low bcl-2/bax ratio, and higher bcl-x _L compared to peripheral blood. Additionally, upon primary stimulation with anti-CD3, cord blood T cell subsets were more resistant to apoptosis compared to peripheral blood. In contrast, rechallenge of previously stimulated lymphocytes with anti-CD3 monoclonal antibody triggered apoptosis in a larger proportion of T cells from cord blood as compared to peripheral blood, whereas the number of T cells undergoing anti-Fas-induced programmed cell death were lower in cord blood compared to peripheral blood.     

锌对于原发性肝细胞癌BEL-7404细胞生物学行为的影响

目的:观察外源锌对原发性肝细胞癌(HCC)BEL-7404细胞生物学行为的影响。方法:应用TSQ锌离子荧光探针、MTT法、DNA倍体法、吖啶橙/溴乙啶双荧光染色法和Transwell小室法分别检测不同浓度硫酸锌刺激下BEL-7404细胞内锌离子含量、细胞活力、细胞周期、细胞凋亡及细胞迁移和侵袭能力的变化。应用real-time PCR和Western blot法分别检测不同浓度锌离子对BEL-7404细胞白蛋白的mRNA和蛋白表达的影响。结果:随着培养环境中锌离子浓度的升高,BEL-7404细胞内的锌离子含量增加,细胞的存活率和细胞迁移与侵袭能力降低,凋亡率升高(P0.05);G0/G1期细胞比例降低,G2/M期细胞比例升高(P0.05);细胞白蛋白的mRNA和蛋白表达量增加(P0.05)。结论:外源性给予锌离子可抑制HCC细胞的活力、迁移与侵袭能力,诱导细胞凋亡,阻滞细胞周期在G2/M期,并可能降低细胞的恶性表型。     

Phenotypical Characterization of Cells in the Thoracic Duct of Patients with and without Systemic Inflammatory Response Syndrome and Multiple Organ Failure

The subset composition and recirculation properties of the migrating lymphocyte pool in humans is largely unknown. The present study was conducted in order to phenotypically characterize cells in human thoracic duct lymph of patients under non-inflammatory and inflammatory conditions. These data were compared with data from peripheral blood, with special emphasis on those cells homing to the gut. Thoracic duct lymph and peripheral blood contained comparable proportions of B and T lymphocytes and CD8⁺ cells. Thoracic duct lymph contained proportionally more CD4⁺ cells, more CD4⁺CD45RO⁺ that express α₄β₇ cells and more CD8⁺CD45RO⁺ that express α₄β₇, as compared to peripheral blood. These data suggest an equal recirculation rate of B and T lymphocytes; a more active recirculation of CD4⁺ cells compared to CD8⁺ cells; and a more active recirculation of memory cells to the gut as compared to other extra-lymphoid sites in patients under non-inflammatory conditions. Data were also obtained in patients with the system inflammatory response syndrome and multiple organ failure. Although it is generally assumed that granulocytes and monocytes do not recirculate, lymph of multiple organ failure patients contained significantly more granulocytes than monocytes, indicating that in severe generalized inflammatory states these cells re-enter the circulation through the thoracic duct. Furthermore, no increased activation of cells homing to the gut was found in these patients.     

Cytokine production and apoptosis among T cells from patients under treatment for Plasmodium falciparum malaria

Available evidence suggests that Plasmodium falciparum malaria causes activation and reallocation of T cells, and that these in vivo primed cells re-emerge into the periphery following drug therapy. Here we have examined the cytokine production capacity and susceptibility to programmed cell death of peripheral T cells during and after the period of antimalarial treatment. A high proportion of peripheral CD3+ cells had an activated phenotype at and shortly after time of admission (day 0) and initiation of therapy. This activation peaked around day 2, and at this time-point peripheral T cells from the patients could be induced to produce cytokines at conditions of limited cytokine response in cells from healthy control donors. Activated CD8hi and TCR-gammadelta+ cells were the primary IFN-gamma producers, whereas CD4+ cells constituted an important source of TNF-alpha. The proportion of apoptotic T cells was elevated at admission and peaked 2 days later, while susceptibility to activation-induced cell death in vitro remained increased for at least 1 week after admission. Taken together, the data are consistent with the concept of malaria-induced reallocation of activated T cells to sites of inflammation, followed by their release back into the peripheral blood where they undergo apoptotic death to re-establish immunological homeostasis as inflammation subsides. However, the high proportion of pre-apoptotic cells from the time of admission suggests that apoptosis also contributes to the low frequency and number of T cells in the peripheral circulation during active disease.     

In Vivo Activation of T-Cell Induction into the Primed Phenotype and Programmed Cell Death by Staphylococcal Enterotoxin B

The authors demonstrate that SEB immunization activates Vβ8⁺ T cells and induces the acquisition of the primed phenotype as defined previously by low MEL-14 and high Pgp-1 expression. SEB-activated spleen CD4⁺ and CD8Vβ8⁺ T cells have different population dynamics and regulate the expression of MEL-14 and Pgp-1 differentially, suggesting that the SEB-MHC class II complex preferentially activates CD4Vβ8⁺ T cells. Interestingly, at day 3 after SEB immunization, Vβ8⁺ T cells expressing low, but not high, levels of MEL-14 undergo apoptosis, indicating that T-cell activation is a prerequisite for triggering programmed cell death. These results might help to trace antigen-reactive cells to the activated or primed pool, as well as to identify those cells which will undergo programmed cell death.     

Immunotoxic Effects of Mercuric Compounds on Human Lymphocytes and Monocytes. II. Alterations in Cell Viability

The major goal of this investigation was to examine the cytotoxic properties of both HgCl₂ and MeHgCl, in terms of their ability to alter human T-cell and monocyte viability. Following treatment with HgCl₂ (0-20μ;g/ml) or MeHgCl (0-2μ;g/ml), there was minimal reduction in lymphocyte viability at 1-4 hr. However, after exposure to mercury for 24 hr, cell death was apparent. In comparison, monocytes exhibited significant loss of viability during the early exposure periods. MeHgCl was approximately 5-10 times more potent than HgCl₂. Other indicators of cell death were also determined. Measurement of the energy charge ratio indicated profound changes in cellular energy conservation. Electron microscopic analysis of cells treated with mercury revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation and condensation of nucleoplasm. In concert with these nuclear changes, there was destruction of cytoplasmic organelles with loss of membrane integrity. Studies of phospholipid synthesis by mercury treated cells confirmed that there were alterations in membrane structure. Thus, there was a decrease in total phosphatide synthesis by treated cells. Moreover, monocyte phospholipid synthesis appeared to be more sensitive to the presence of mercury then lymphocytes. Finally, both forms of mercury caused a rapid and sustained elevation in the intracellular levels of Ca⁺⁺. These morphological and biochemical changes are consistent with the notion that mercury initiates cytotoxic changes associated with programmed cell death.     

Induction of apoptosis in monocytes by Mycobacterium leprae in vitro: a possible role for tumour necrosis factor-alpha

A diverse range of infectious organisms, including mycobacteria, have been reported to induce cell death in vivo and in vitro. Although morphological features of apoptosis have been identified in leprosy lesions, it has not yet been determined whether Mycobacterium leprae modulates programmed cell death. For that purpose, peripheral blood mononuclear cells obtained from leprosy patients were stimulated with different concentrations of this pathogen. Following analysis by flow cytometry on 7AAD/CD14+ cells, it was observed that M. leprae induced apoptosis of monocyte-derived macrophages in a dose-dependent manner in both leprosy patients and healthy individuals, but still with lower efficiency as compared to M. tuberculosis. Expression of tumour necrosis factor-alpha (TNF-alpha), Bax-alpha, Bak mRNA and TNF-alpha protein was also detected in these cultures; in addition, an enhancement in the rate of apoptotic cells (and of TNF-alpha release) was noted when interferon-gamma was added to the wells. On the other hand, incubation of the cells with pentoxifylline impaired mycobacterium-induced cell death, the secretion of TNF-alpha, and gene expression in vitro. In addition, diminished bacterial entry decreased both TNF-alpha levels and the death of CD14+ cells, albeit to a different extent. When investigating leprosy reactions, an enhanced rate of spontaneous apoptosis was detected as compared to the unreactive lepromatous patients. The results demonstrated that M. leprae can lead to apoptosis of macrophages through a mechanism that could be at least partially related to the expression of pro-apoptotic members of the Bcl-2 protein family and of TNF-alpha. Moreover, while phagocytosis may be necessary, it seems not to be crucial to the induction of cell death by the mycobacteria.     

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.     

Apoptosis and HIV infection: T-cells fiddle while the immune system burns.

Enhancement of programmed cell death (apoptosis) of CD4 T-cells by human immunodeficiency virus (HIV) is thought to be an important factor in the pathogenesis of HIV disease. Recent studies have cast doubt on this concept, however, portraying apoptosis as a potent antiviral strategy to eliminate infected cells. These studies have shed new light on the role of apoptosis in HIV infection. While cellular and immunologic mechanisms of apoptosis purge the HIV-infected lymphoid cell population, HIV thwarts apoptosis in myeloid cells, particularly monocyte/macrophages. Although HIV protease inhibitor therapy partially reverses the lymphoid cell process, this therapeutic approach fails to counter the persistence of HIV infection in myeloid cells. Thus apoptosis of T-cells may be a futile host attempt to control the spread of HIV while the infection smoulders in monocyte/macrophages. In other words, the antiviral defense system fiddles while the immune system burns.     

Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.