Caspase-independent cell death in T lymphocytes

T lymphocyte death is essential for proper function of the immune system. During the decline of an immune response, most of the activated T cells die. Cell death is also responsible for eliminating autoreactive lymphocytes. Although recent studies have focused on caspase-dependent apoptotic signals, much evidence now shows that caspase- independent, necrotic cell death pathways are as important. An understanding of the molecular control of these alternative pathways is beginning to emerge. Damage of organelles including mitochondria, endoplasmic reticulum or lysozymes, leading to an increase in calcium and reactive oxygen species and the release of effector proteins, is frequently involved in caspase-independent cell death.     

Influence of the 3D-conformation,glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen. Use of recombinant isoforms and specific monoclonal antibodies as immunological tools

Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia pastoris by recombinant methods, have been used as tools to determine the role of the three-dimensional (3D)-folding, the glycan component and several point changes of the amino acid sequence in the binding of murine IgG mAbs and human IgE to the olive allergen. Seven mAb families (F1-F7) were established, two of which (F1 and F2) recognize continuous epitopes. The carbohydrate moiety of Ole e 1 was involved in the binding to F2 and F4, whereas F3 and F7 were able to bind to all Ole e 1 variants. The remaining families of IgG murine antibodies exhibited different affinities for the antigens assayed in a native or denatured conformation. Although the binding of human IgE to Ole e 1 was not affected by heat treatment, it was shown to be strongly dependent on the integrity of the disulfide bridges and was partially inhibited by F3-F7 IgG antibodies, their individual values ranging from 12 to 31% and reaching 53% with their mixture. The IgE from sera of olive-allergic patients showed a significant diversity of binding capacity to the members of the Ole e 1-like family due to the microheterogeneity of their polypeptide sequences, in spite of their highly conserved primary structures. Whereas one of the isoforms of Ole e 1 exhibits a highly similar behavior to the natural form, being a putative molecule for diagnostic purposes, other ones can be considered as hypoallergenic variants of this allergen and, thus, potential candidates to be used in immunotherapy.     

Programmed cell death: many ways for cells to die decently

Apoptosis, a cell death programme mediated by the caspase family of cysteine proteases, is essential for appropriate removal of excess cells in many developmental and physiological settings. It would, however, be very dangerous for the organism to depend on a single protease family for clearance of unwanted and potentially dangerous cells. Indeed, the exclusive role of caspases in the execution of programmed cell death (PCD) has been challenged recently, and the understanding of the molecular control of alternative death pathways is emerging. Here, I review recently discovered triggers and molecular regulators of caspase-independent cell death programmes and discuss their potential as therapeutic targets for the treatment of cancer.     

Laboratory and field testing of particle size-selective sampling methods for mineral dusts

The performances of eight sampling devices were tested with mineral dusts in the laboratory and in a talc production plant. The IOM sampler was chosen as the reference method for inhalable dust, and the IOM samplers provided with the porous plastic foam media were used as the reference methods for both the thoracic and respirable aerosols. The other size-selective instruments tested included the Respicon virtual impactor, the optical GRIMM aerosol monitor, and a two-stage cascade impactor with cut points of 10 and 4 microm. The 37-mm cassettes were also included both as open- and closed-face versions. The study confirmed the usability of the IOM samplers for mineral dust, not only in its original version for the inhalable fraction but also its modified versions for the thoracic and respirable fractions. A high correlation with the two-stage impactor results is an indication of good reproducibility. The results increased the evidence that the 37-mm cassette is a poor indicator of inhalable aerosol. The concentrations obtained with both cassette methods were not only systematically too low but also showed large collection efficiency variability. Therefore, the results cannot be corrected by using correction factors. The concentrations of inhalable aerosol measured with the Respicon were generally low, but its performances for the thoracic and respirable fractions were closer to those for the reference samplers. The results also indicate that the GRIMM monitor is well-suited for such mineral dust determinations when very good accuracy is not required, but the immediate availability of the result is more important.     

Age-related liquefaction of the human vitreous body: LM and TEM evaluation of the role of proteoglycans and collagen

PURPOSE: To evaluate morphologic aspects of age-related liquefaction of the human vitreous body by light and electron microscopy to provide a basis from which future studies directed at the pathogenesis of this phenomenon can be undertaken. The study focuses on changes in fibrillar collagen and proteoglycans (PGs). METHODS: Morphologic aspects of intravitreal liquefied spaces and matrix areas surrounding them were examined in 13 adult human donor eyes (aged 21-80 years) by light (LM) and transmission electron microscopy (TEM). Collagen fibrils were visualized by using standard contrasting methods. PGs were specifically stained by cupromeronic blue (CB). RESULTS: Eyes from older donors contained larger spaces than eyes from younger ones. Transitions between matrix and spaces were abrupt or gradual. In transition areas of all specimens, a gradual decrease in the number of collagen fibers, and to a lesser extent of PGs was observed. In addition, a fragmentation of collagen fibers and an aggregation of PG-molecules around these fragments were found. Neither cells nor their fragments were observed in these areas. CONCLUSIONS: This is the first study to evaluate vitreous liquefaction at the light and electron microscopic level. A breakdown of collagen fibrils into smaller fragments seems to be crucial to the pathogenesis of age-related liquefaction of the human vitreous body. The mechanism inducing fragmentation of vitreous fibrils has yet to be elucidated. From the absence of cells and cellular remnants in all specimens, it is tentatively concluded that an extracellular process is involved.     

Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors

Asymmetric dimethylarginine (ADMA) and monomethylarginine (LMMA) are endogenous inhibitors of nitric oxide synthase. A high level of ADMA in plasma has shown to be a significant risk factor for acute coronary syndromes and elevated plasma ADMA levels are prevalent in patients with hypercholesterolemia. It was therefore hypothesized that lowering plasma cholesterol levels with statin treatment would also lower ADMA concentrations. This double-blind study addressed the effect of high-dose statin treatment on plasma levels of ADMA and LMMA. Forty-eight subjects with mild hypercholesterolemia were randomly assigned to receive simvastatin 80 mg/d, atorvastatin 40 mg/d, or placebo for 8 weeks. Both statins decreased low-density lipoprotein cholesterol effectively (simvastatin 54% and atorvastatin 49%). However, concentrations of arginine derivatives remained unchanged during statin treatment and did not correlate with cholesterol levels. This study indicates that statin treatment has no clear influence on plasma ADMA or LMMA concentrations.     

Reproduction,blood and plasma parameters and gill histology of vendace (Coregonus albula L.) in long-term exposure to acidity and aluminum

Vendace were exposed to pHs 4.75 and 5.25 with or without added aluminum (200 microg=7.4 micromol AlL(-1)) from late endogenous vitellogenesis in July through the spawning period. At the normal time of spawning, when 48% of the control females had already released their eggs, 50% of females at pH 4.75+Al had completely unovulated oocytes. The final proportions of completely ovulated females were 14%, 36%, 25%, 61%, and 81% at pH 4.75+Al, pH 4.75, pH 5.25+Al, pH 5.25, and in the control group, respectively. Delayed testes regression was seen in males at pH 4.75+Al. A clear decrease in plasma Na(+) and Cl(-) and an increase in blood glucose concentration was detected only near spawning time, from October to November, coincident with Al accumulation inside the gill tissue. It is concluded that seasonal changes, probably related to reproductive physiology or to the decrease in water temperature, are associated with the increase in Al toxicity in vendace.     

Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant

Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.     

Biologic and therapeutic role of HER2 in cancer

Overexpression of the human epidermal growth factor-2 (HER2) oncogene in human breast carcinomas has been associated with a more aggressive course of disease. The reason for this association is still unclear, although it has been suggested to rest in increased proliferation, vessel formation, and/or invasiveness. Alternatively, prognosis may not be directly related to the presence of the oncoprotein on the cell membrane, but instead to the breast carcinoma subset identified by HER2 overexpression and characterized by a peculiar gene expression profile. HER2 has also been associated with sensitivity to anthracyclins and resistance to endocrine therapy, suggesting that tyrosine kinase receptor and hormone receptor pathways represent two major proliferation pathways exclusively active in breast carcinomas, one sensitive to chemotherapeutic drugs and the other to antiestrogens. HER2 currently represents one of the most appropriate targets for specific therapy. Indeed, trastuzumab, a monoclonal antibody directed against the extracellular domain of HER2, is therapeutically active in HER2-positive breast carcinomas. However, a consistent number of HER2-positive tumors is not responsive to HER2-driven therapy, indicating the need for a better understanding of the mechanism of action of this new biological drug in vivo. While preclinical studies suggest antibody-dependent cell cytotoxicity as the major mechanism, determination of NK activity at the time of treatment remains mandatory, especially in patients treated with immunosuppressive drugs. The efficacy of prophylactic vaccination has been fully demonstrated in preclinical models, whereas ongoing studies of active immunotherapy using a variety of vaccination regimens against HER2 in tumor-bearing mice and patients have met with only moderate success.