Protective effects of glutamine on human melanocyte oxidative stress model.

BACKGROUND: Vitiligo is a disorder caused by the loss of the melanocyte activity on melanin pigment generation. Studies show that oxidative-stress induced apoptosis in melanocytes is closely related to the pathogenesis of vitiligo. Glutamine is a well known antioxidant with anti-apoptotic effects, and is used in a variety of diseases. However, it is unclear whether glutamine has an antioxidant or anti-apoptotic effect on melanocytes. AIMS: The aim of this study was to investigate the protective effects of glutamine on a human melanocyte oxidative stress model. METHODS: The oxidative stress model was established on human melanocytes using hydrogen peroxide. The morphology and viability of melanocytes, levels of oxidants [reactive oxygen species and malondialdehyde], levels of antioxidants [superoxide dismutase and glutathione-S-transferase], and apoptosis-related indicators (caspase-3, bax and bcl-2) were examined after glutamine exposure at various concentrations. Expressions of nuclear factor-E2-related factor 2, heme oxygenase-1, and heat shock protein 70 were detected using western blot technique after glutamine exposure at various concentrations. RESULTS: Our results demonstrate that pre-treatment and post-treatment with glutamine promoted melanocyte viability, increased levels of superoxide dismutase, glutathione-S-transferase and bcl-2, decreased levels of reactive oxygen species, malondialdehyde, bax and caspase-3, and enhanced nuclear factor-E2-related factor 2, heme oxygenase-1, and heat shock protein 70 expression in a dose dependent manner. The effect of pre-treatment was more significant than post-treatment, at the same concentration. LIMITATIONS: The mechanisms of glutamine activated nuclear factor-E2-related factor 2 antioxidant responsive element signaling pathway need further investigation. CONCLUSIONS: Glutamine enhances the antioxidant and anti-apoptotic capabilities of melanocytes and protects them against oxidative stress.

Why does tuberculosis lead to specific inflammation?

When Mycobacterium tuberculosis infects humans, about 20% of those infected actually develop tuberculosis (TB). In Japan, the incidence of TB in 2008 was 24,760 cases (19.4/100,000 persons) and the rate has been decreasing gradually, but is still higher than in the USA, Holland, and Belgium, for example. Histologically, tuberculosis displays exudative inflammation, proliferative inflammation and productive inflammation depending on the time course. In productive inflammation, granulomatous lesions with necrotic centers are formed. The typical granulomas consist of epithelioid macrophages, Langhans' multinucleated giant cells, lymphocytes and fibroblasts, and the process of their formation involves many cytokines, chemokines and transcription factors. These findings have been derived primarily from animal experiments utilizing an airborne infection apparatus. The conditions for airborne infection have been described in detail elsewhere. This mini-review focuses on what has been found through animal experiments, and also indicates areas for which data are not currently available.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae.

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

Rifampicin protects PC12 cells against MPP+-induced apoptosis and inhibits the expression of an alpha-Synuclein multimer.

The potential cytoprotective effects of the anti-leprosy antibiotic rifampicin were investigated in rat pheochromocytoma (PC12) cells prior to intoxication with 1-Methyl-4-phenyl pyridinium (MPP(+)). MPP(+) induced both apoptotic and necrotic cell death, and increased the expression of a 57 kDa species of alpha-Synuclein. This species of alpha-Synuclein is larger than the monomer, and is therefore an oligomer or an aggregated form of the protein. Rifampicin significantly increased survival of these catecholaminergic cells in a concentration-dependent manner. The expression of the higher molecular mass alpha-Synuclein was increased by MPP(+) exposure, and its expression was inversely related to cell survival in the rifampicin-treated cells. Importantly, rifampicin suppressed apoptosis almost completely, without shifting the death cascade to necrosis, which is a problem that has been reported with caspase inhibitors of apoptosis (Hartmann, A., Troadec, J.D., Hunot, S., Kikly, K., Faucheux, B.A., Mouatt-Prigent, A., Ruberg, M. Agid, Y., Hirsch, E.C., 2001. Caspase-8 is an effector in apoptotic death of dopaminergic neurons in Parkinson's disease, but pathway inhibition results in neuronal necrosis. J. Neurosci. 21, 2247-2255). These results suggest that rifampicin improves survival of catecholamine- and alpha-Synuclein-containing cells, which degenerate in Parkinson's disease (PD), and thus may be therapeutic in this disease.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression.

BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii.

Haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, were investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated haemocytes of M. rosenbergii were cultured at a ratio of 1:50 haemocytes to pathogen with the yeast Debaryomyces hansenii, the bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25 degrees C for 2 h, followed by washing to remove free particles. At least 200 haemocytes were counted to determine the phagocytosis rate, and the results showed that haemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing haemocytes displayed morphological changes characteristic of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation and fragmentation of nuclei. This pathogen-induced apoptosis was further confirmed by DNA laddering and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labelling) assays. Neither haemocytes treated with latex beads nor uninfected haemocytes (control group) showed signs of apoptosis after 48 h in culture.

Cytokines and Mycobacterium leprae induce apoptosis in human Schwann cells.

The development of deformities during the course of leprosy disease is a major public health concern worldwide. It is possible that cytokine production and apoptosis of Schwann cells (SCs) directly affect nerve degeneration and regeneration leading to injury of the myelin sheath and axon. In the present study, the expression of TNFalpha, TGFbeta, and their receptors, in addition to cell death triggered by cytokines or whole Mycobacterium leprae were investigated in a human SC line. The results showed the presence of TNF-Rs and TGF-RII on the SC membrane and the shedding of TNF-Rs during the culture period. Evaluation of cell death was performed through TUNEL and flow cytometry techniques. TNFalpha/TGFbeta combination as well as M. leprae infection triggered an increase in the apoptosis rate in the cultured SC. Moreover, reverse transcriptase-polymerase chain reaction assay revealed that M. leprae upregulated the expression of such cytokines and their receptors on the SC line. Despite the detection of TNFalpha mRNA, no protein was found in the culture supernatants. The data indicate that induction of SC death after cell interaction with M. leprae may, in fact, be implicated in the pathogenesis of nerve damage, which can most likely be modulated by in vivo cytokine production.

Tunel and limited immunophenotypic analysis of apoptosis in paucibacillary leprosy lesions

Some mycobacterial infections, such as tuberculosis, are characterized by apoptosis in infected or by-stander mononuclear immune cells. For localized (paucibacillary, PB) and diseminated (multibacillary, MB) leprosy, characterized by polarized Thl-like vs, Th2-like immune responses, respectivelly little is known about lesional apoptosis. We analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Some TUNEL (+) PB sections were then reacted with phycoerythein-conjugated (red)antibodies against T cells, monocytes, or antigen-presenting (Langerhans) cells. TUNEL (+) bodies were detected in 9 of 16 PB lesions (56%) and in 1 of 5 MB lesions (20%). Some TUNELL (+) bodies in PB disease were CD3+ (T cell), as well as CD4+ (T-helper) or cd8+ (T-cytotoxic) Apoptosis characterizes PB and MB leprosy lesions and may be more freqeunt in PB disease. In PB disease, some TUNEL (+) bodies may derive from T cells

AIF: a multifunctional cog in the life and death machine.

Mitochondria have a dual role in cellular life and death as life-promoting energy providers and as contributors to programmed cell death (apoptosis). The precise sequence of events resulting in the permeabilization of the mitochondrial membrane and the release of mitochondrial resident proteins remains an actively explored topic. Hansen and Nagley describe results from mammalian cells and from the nematode C. elegans that lead to a feedforward model for mitochondrial destabilization. Furthermore, they describe the mitochondrial and apoptotic functions of several proteins released from mitochondria during progression toward cell death.

Modulation of p53 dependent gene expression and cell death through thioredoxin-thioredoxin reductase by the Interferon-Retinoid combination.

We have shown earlier that the IFN-beta and all-trans retinoic acid (RA) combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic technique we have identified several Genes associated with Retinoid-IFN induced Mortality (GRIM). One of the GRIMs was human thioredoxin reductase (TR), a redox enzyme. Since the overexpressed TR augments IFN/RA stimulated cell death, we explored the mechanisms of TR-mediated death. Here we show that TR augments cell death by upregulating the transcriptional activity of p53 tumor suppressor. This process does not involve a physical increase in levels of p53. Using redox inactive mutants of TR and its substrate, thioredoxin (Trx), we demonstrate that IFN/RA-induced regulation of p53 dependent gene expression requires TR and Trx. In contrast-over-expression of wildtype TR or Trx augment the p53 dependent gene expression in response to IFN/RA treatment. Consistent with these results an increased DNA binding activity of p53 was noted in the presence of TR. These studies identify a novel mechanism of p53 mediated cell death regulation involving redox enzymes.