An early oxygen-dependent step is required for dexamethasone-induced apoptosis of immature mouse thymocytes.

The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.

Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.

Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1 beta converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4(+) and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.

Oxidative protein damage and degradation in lymphocytes from patients infected with human immunodeficiency virus.

It has been proposed that oxidative stress is the common mediator of apoptotic cell death in AIDS. However, mechanistic relationships between oxidative damage and cell death are far from clear. It is reported here that the mitogenic activation of T lymphocytes from human immunodeficiency virus-positive subjects involves perturbation of redox balance, as indicated by the increase in hydroethydine intracellular oxidation and manganese superoxide dismutase adaptive induction. Principal molecular targets of oxidative injury are cellular proteins whose content in carbonyl groups increases together with a dramatic increase in degradation of newly synthesized proteins catalyzed by the ATP- and ubiquitin-dependent proteolytic system. The major consequence of this metabolic anomaly is the decrease in protein cell mass leading to cells that are smaller than normal at lethal mitosis.

HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals.

T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.

The early intracellular production of a reactive oxygen intermediate mediates apoptosis in dexamethasone-treated thymocytes.

Many conditions that induce an oxidative stress are capable of evoking apoptosis. This has lead to the proposal of oxidative stress as a mediator of apoptosis. We show that, in murine thymocytes, oxidative stress and apoptosis occur in the same cell. We identified four distinct apoptotic subpopulations that appeared sequentially in time. Catalase protected from dex-amethasone-induced death in the initial stages of apoptosis, while iron chelators and Vitamin E did not. Further studies provided evidence supporting the early production of an intracellular oxidative intermediate as an obligatory step for the efficient induction of apoptosis. We propose that at least one of the molecules capable of filling this role is hydrogen peroxide.

[Evaluation of clinical and humoral data in 148 patients with Piaget's bone disease]. / Osteitis deformante de Paget. Manifestaciones clínicas y humorales en 148 pacientes.

Pain was present in 113 out of a total of 148 patients with Paget's bone disease who were evaluated. The symptom was originated by: a) bone, in 56.6 % of the cases; b) joints, in 32.7 %; c) skull compression in 7.1 % and spine compression in 3.5 % of the cases. Bone deformities were seen in 50.7 % of patients. Of this total, 41.4 % were localized in femorae. Pathological fractures were found in 16.9 % of patients. Of this total, 44.0 % were localized in femore and 19.2 % in the humerae. The localization of Paget's bone disease in the humerus appears to be specially risky due to the high percentage of fractures found in the present study as well as the frequency of the sarcomatous degeneration described in the literature. Two patients (1.4 %) developed an osteogenic sarcoma in bones previously affected by Paget's disease. It was localized in the tibia in one case and in iliac bone in the other. Frequent associated diseases were osteoporosis, verified in 31.8 % of the patients, arthritis deformans in 28.6 % and nephrolithiasis, verified in 31.8 % of the patients, arthritis deformans in 28.6 % and nephrolithiasis in 12.2 %. Associated neoplastic diseases were mammary cancer in 6.5 % of 77 women and colonic cancer in 2.0 of 148 patients of both sexes. The diagnosis has been made previously or during the period of the present study. The effects of the extent of Paget's bone disease as well as the effect of Paget's skull complication on serum alkaline phosphatase and urinary total hydroxyproline were investigated. Serum alkaline phosphatase and urinary total hydroxyproline were higher in polyostotic then in oligostotic patients although the difference was not significant for serum alkaline phosphatase in a group of 50 polyostotic with skull lesions versus 16 oligostotic with skull lesions. There was a significant increase of serum alkaline phosphatase in groups of 29 polyostotic without skull lesions versus 45 oligostotic without skull lesions (p < 0.001) and in groups of 50 polyostotics with skull lesions versus 29 polyostotics with skull lesions (p < 0.05), and of 16 oligostotic withh skull lesions versus 45 oligostotics without skull lesions (p < 0.02). There was a significant increase of urinary total hydroxyproline in a group of 46 polyostotics with skull lesions versus 15 oligostotics with skull lesions (p < 0.01) and of 25 polyostotics without skull lesions versus 41 oligostotics without skull lesions (p < 0.001), as well of 46 polyostotics with skull lesions versus 25 polyostotics without skull lesions (p < 0.005) and 15 oligostics with skull lesions versus 41 oligostotics without skull lesions (p < 0.02). Although the differences should be referred to a greater number of patients, the results suggest that the effects of Paget skull lesions are more evident on serum alkaline phosphatase than on urinary total hydroxyproline.

Pycnodysostosis: some aspects concerning kinetics of calcium metabolism and bone pathology.

A 23-year-old white woman suffering pycnodysostosis whose parents and five siblings were unaffected was investigated. Chromosomal morphology was normal. Histologic examination of a biopsy specimen obtained from the distal phalanx of the left thumb, corresponding to an area on an x-ray film of osteolysis, showed a fibrous dysplasia-like picture. Kinetic studies of calcium metabolism revealed that exchangeable pool size, turnover, and bone accretion rate were all decreased. Intestinal calcium absorption was investigated simultaneously by a double isotope technique and by deconvolution of the plasmatic specific activity curve of 47Ca given per os. Results obtained by both methods coincided in that values were found to be similarly increased. Endogenous fecal calcium was also determined and revealed a similar increase. Some physiopathological implications of these findings are discussed.