Systematic investigation of different steroid precursors with respect to their effect on superoxide anion production by human neutrophil granulocytes.

Free radicals are involved in several pathological processes in living organisms, for example in athero- and oncogenesis. Some steroids are known to be effective antioxidants, while others do not play any such role. The aim of our study was to examine the antioxidant capability of different metabolites in the synthesis of steroid hormones. As a model, we chose human neutrophils producing superoxide anion, which is the source of many other radicals. Neutrophils were separated from healthy volunteers. Isolated cells were incubated with varying concentrations of steroid compounds and stimulated with N-formyl-Met-Leu-Phe. Superoxide anion production was determined by photometry. Neutrophils incubated with corticosterone and 18-hydroxy-deoxycorticosterone showed a significant reduction in superoxide production, whereas we found a significant enhancement in the presence of 11beta-hydroxyprogesterone. Furthermore, we observed a non-significant decreasing trend after incubation with cholesterol 3-sulphate and an increasing tendency using 11-hydroxyandrostenedione. We were also able to produce newer morphological and functional evidence of the role of myeloperoxidase enzyme in the steroidal antioxidant effect by electronic microscopy and use of sodium hypochlorite in our incubation model. Based on these results, we conclude that not only steroid end products but also their intermediate metabolites, most of which are also present in human plasma, partly influence free radical metabolism. Thus, this study provides further argument for the search for the molecular basis responsible for the antioxidant effect of steroid structures. This may lead to new opportunities for finding really efficient antioxidants, which might perhaps be used in a combined manner with other agents in the fight against certain life-threatening diseases.

Inhibition of C1q-beta-amyloid binding protects hippocampal cells against complement mediated toxicity.

Activation of complement by beta-amyloid (A beta) contributes to the pathology of Alzheimer's disease (AD). Here, we show that C1-Inhibitor (C1-Inh) protects cultured rat hippocampal cells against beta-amyloid induced complement lysis indicating a classical pathway-mediated activation mechanism. We report on screening of compound libraries to identify compounds that inhibit C1q binding to beta-amyloid. Characterization of these compounds revealed that C1q possessed distinct binding sites for beta-amyloid and antibodies. One selected compound protected cultured hippocampal cells against complement-dependent beta-amyloid toxicity. These results provide evidence that complement has the potential to damage hippocampal cells, and C1q is a relevant target to suspend this deleterious mechanism in Alzheimer's disease.

Induced myeloperoxidase activity and related superoxide inhibition during hormone replacement therapy.

OBJECTIVE: To test whether the menopause entails any changes in the myeloperoxidase activity of neutrophil granulocytes. The effects of hormone replacement therapy on myeloperoxidase activity and related changes in free radical production were also investigated. DESIGN: Laboratory investigation of the effect of oestrogen on intracellular myeloperoxidase activity and release from human neutrophil granulocytes. Analysis of related changes in superoxide anion generation. SETTING: 2nd Department of Medicine and 1st Department of Obstetrics and Gynaecology, Semmelweis University, Budapest. SAMPLES: Intracellular myeloperoxidase activity (mean peroxidase index) was measured automatically in blood samples obtained for general laboratory work-up from 135 randomly selected patients in our department. Blood samples from 11 postmenopausal women were analysed before and during hormone replacement therapy. Blood samples from 20 healthy volunteers were obtained and neutrophil granulocytes separated for in vitro measurement of superoxide anion production after adding myeloperoxidase to the incubation media. METHODS: The mean peroxidase index was measured using a Technicon H-3 instrument. myeloperoxidase release from neutrophils was quantified by ELISA technique. Superoxide production of isolated neutrophil granulocytes was measured by photometry. MAIN OUTCOME MEASURES: Intracellular activity of myeloperoxidase, concentration of myeloperoxidase-protein in supernatant of neutrophils, release of superoxide anion from neutrophil granulocytes. RESULTS: 1. Intracellular myeloperoxidase activity in neutrophils was lower in postmenopausal women, than in females with regular cycles (-1.84 +/- 3.06 versus 1.59 +/- 3.55, P < 0,001). 2. In postmenopausal women intracellular myeloperoxidase activity and myeloperoxidase release increased during hormone replacement therapy (-5.54 +/- 6.63 versus -0.2 +/- 6.05; P < 0.001 and 52.74 mU/ml +/- 25.73 versus 251.4 +/-234.1 mU/ml; P < 0.05). 3. Adding myeloperoxidase to neutrophil granulocyte suspensions, the production of superoxide anion fell (e.g. adding 280 ng/ml myeloperoxidase: 77.9 +/- 14.04 % of control production, P < 0.001). CONCLUSION: Hormone replacement restores the reduced myeloperoxidase activity in menopausal women. Adding myeloperoxidase to neutrophil granulocytes, the production of free radicals decreases.

Effect of transforming growth factor-beta1 on microglial MHC-class II expression.

In the present report, the effects of IFN-gamma and transforming growth factor beta1 (TGF-beta1) on major histocompatibility complex class II (MHC-II) gene expression in isolated mouse brain microglial cells, in the MH-S macrophage cell line and in the primary mouse macrophage cultures were examined. IFN-gamma is a potent inducer of MHC-II gene and this induction was further elevated in microglia by TGF-beta1, while TGF-beta1 inhibited IFN-gamma, induction in macrophages. The enhancing effect of TGF-beta1 was also detected in microglia at the protein level. Transient transfection of microglia with 5' deletional mutants of the MHC-II IAalpha promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated that TGF-beta1 acts at the transcriptional level to enhance the MHC-II expression induced by IFN-gamma.

Differential regulation of major histocompatibility complex class II expression and nitric oxide release by beta-amyloid in rat astrocyte and microglia.

Astrocytes and microglial cells were examined for expression of two immunologically important molecules, major histocompatibility complex class II (MHC-II) and nitric oxide (NO) following treatment with IFN-gamma and beta-amyloid (betaA) peptides, betaA(1-42) and betaA(25-35). IFN-gamma is a potent inducer of both MHC-II gene expression and NO production. The induction of MHC-II was inhibited by both betaA peptides in astrocytes but they had little or no effect in microglia. betaA peptides had no effect on NO release in astrocytes but on microglia betaA(1-42) synergistically induced NO release with IFN-gamma. Transient transfection of astrocytes with 5' deletional mutants of MHC-II IAalpha promoter linked to the chloramphenicol acetyl transferase reporter gene (IAalpha-CAT), demonstrated that betaA acts at the transcriptional level to downregulate IFN-gamma induced MHC-II gene expression in astrocytes. In previous studies, the induction of MHC-II on glial cells were suggested to be involved in the pathogenesis of neurodegenerative diseases and MHC-II(+) microglial cells were observed at much higher frequency than astrocytes. This study provides information on the regulation of the MHC-II gene expression in astrocytes and in microglial cells by betaA and this pathway may be critically involved in the immune/inflammatory regulation within the central nervous system.

Repression of MHC class II gene transcription in trophoblast cells by novel single-stranded DNA binding proteins.

The maintenance of the fetus during pregnancy has been attributed to the absence of major histocompatibility complex (MHC) class II antigens on fetal trophoblastic cells that make contact with the maternal immune system. However, the mechanism(s) by which class II genes are regulated in trophoblast cells is unclear. We have identified a negative regulatory element (IA alpha NRE) in the promoter of the mouse class II gene IA alpha that represses IA alpha transcription in trophoblast cells. IA alpha NRE, located from-839 to -828, binds transacting factors from rat, mouse and human trophoblast cells, but not from 18 other cell lines tested. These results indicate that IA alpha NRE binding proteins (IA alpha NRE BPs) are conserved in species with hemochordial placentas, and suggest that IA alpha NRE binding activity is restricted primarily to trophoblast cells. Interestingly, the IA alpha NRE BPs bind to the IA alpha NRE antisense strand in a sequence-specific manner. IA alpha NRE represses transcription from the IA alpha promoter in a position-dependent manner, and has a minor down-regulatory effect on the activity of the SV40 promoter/enhancer. Our results demonstrate that MHC class II gene transcription is repressed in fetal trophoblast cells by sequence-specific, single-stranded DNA binding proteins, and suggest a possible mechanism by which the conceptus is protected from immune rejection during pregnancy.

Activation of multiple transcription factors and fos and jun gene family expression in cells exposed to a single electric pulse.

We report that exposure of cells to a single electric pulse (250-1250 V/cm) results in the rapid and persistent activation of the DNA binding activities of a number of transcription factors, including AP-1, SP1, AP-2, and NF-kappa B, and the transient expression of select members of the fos and jun gene families. Induction of gene expression occurs primarily at the level of transcription, although c-jun expression also appears to be regulated posttranscriptionally. Interestingly, maximal induction of gene expression is detected at electrical field strengths that do not result in pore formation in the plasma membrane and that do not significantly affect cell viability. Exposure of cells to electric pulses does not result in the activation of HSF1 DNA binding activity, or the induction of hsp70 or p53 protein synthesis, indicating that the induction of fos and jun gene expression is not coincident with protein or DNA damage. The results of these studies suggest that electrical pulses may represent a novel mechanism for inducing the activities of multiple transcription factors and the expression of select members of the fos and jun gene families.

Humoral leukocyte adherence inhibition (H-LAI) follow-up trials on malignant process in mice.

Humoral antitumor response of Lewis lung tumor-bearing mice was measured by humoral leukocyte adherence inhibition (H-LAI) test. The reactivity of serum against tumor antigens prepared from primary tumor and lung metastases could be revealed at the first day after injection of tumor cells. On the other hand, the macroscopical appearance of the primary tumors and lung metastases was observed after 5 and 11 days, respectively. These findings suggest that the immune reaction of the host could be detected by H-LAI test earlier than the tumor can manifest itself. The antigens prepared from primary tumors and metastases seemed to be very similar, however, the metastasis antigen had additional determinants as detected by the H-LAI technique. Comparison of two tumor lines, the original Lewis lung tumor (LLT) and its variant with high metastatic capacity (LLT-HH), showed high similarity between their antigens as tested in H-LAI system by cross-reaction probes.

No evidence for human T-cell leukemia virus type I or human T-cell leukemia virus type II infection in patients with multiple sclerosis.

The involvement of human T-cell leukemia viruses (HTLVs) in the pathogenesis of 18 Hungarian patients with multiple sclerosis was investigated. No antibody to HTLVs could be detected in any of the patients. Furthermore, using polymerase chain reaction under highly sensitive conditions, neither HTLV-I DNA nor HTLV-II DNA could be noted in peripheral blood lymphocytes of the patients. Our data do not support a causal association of HTLV-I or HTLV-II with multiple sclerosis.

The effect of 2.45 GHz microwave irradiation on human peripheral lymphocytes.

The effect of 2.45 GHz frequency and 10 mW/cm2 power density microwave irradiation on the in vitro cultured human peripheral lymphocytes blast transformation was examined. In the number of the blast cells we did not find significant difference at cultures irradiated for 1 x 5 hours and 3 x 1 hours as compared to the controls. The number of the blast cells was found to increase when Phytohemagglutinin-stimulated lymphocyte cultures were exposed to repeated irradiation for 6 days (1 hour a day). The microwave irradiation did not influence the degree of the spontaneous blast transformation in any groups.