Genotipificación de HLA-B en pacientes colombianos afectados por el síndrome Stevens-Johnson y la Necrólisis Epidérmica Tóxica

Las reacciones alérgicas a medicamentos cutáneas severas (RAM) como el Síndrome Stevens Johnson (SJS) y la Necrólisis Epidérmica Tóxica (NET),caracterizadas por exantema, erosión de la piel y las membranas mucosas, flictenas, desprendimiento de la piel secundario a la muerte de queratinocitos y compromiso ocular. Son infrecuentes en la población pero con elevada morbi-mortalidad, se presentan luego de la administración de diferentes fármacos. En Asia se ha asociado el alelo HLA-B*15:02 como marcador genético para SJS. En Colombia no hay datos de la incidencia de estas RAM, ni de la relación con medicamentos específicos o potenciales y tampoco estudios de aproximación genómica de genes de susceptibilidad.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases

Background Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. Aims To evaluate expression of death receptors` family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. Subjects and Methods Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-Delta Delta Ct). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. Results In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. Conclusions The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.

Alteração da expressão de sFAS e sFASL na leishmaniose voisceral canina: Juliana Perosso Borges. -

Pós-graduação em Ciência Animal - FMVA

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rolle der FOS-Proteine und des MAPK-Signallings in der Regulation der zellulären Antwort auf genotoxischen Stress

Die mittlere Überlebenszeit nach Erkennung eines Glioblastoms ohne Behandlung liegt bei 3 Monaten und kann durch die Behandlung mit Temozolomid (TMZ) auf etwa 15 Monate gesteigert werden. Neben TMZ sind die chlorethylierenden Nitrosoharnstoffe die meistversprechendsten und am häufigsten eingesetzten Chemotherapeutika in der Gliomtherapie. Hier liegt die mittlere Überlebenszeit bei 17,3 Monaten. Um die Therapie des Glioblastoms noch effektiver zu gestalten und Resistenzen zu begegnen, werden unterschiedlichste Ansätze untersucht. Eine zentrale Rolle spielen hierbei das activator protein 1 (AP-1) und die mitogen aktivierten Proteinkinasen (MAPK), deren Funktion in bisherigen Arbeiten noch unzureichend beleuchtet wurde.rnBesonders mit der Rolle des AP-1-bildenden Proteins FRA-1 in der Therapie des Glioblastoms haben sich bisher nur wenige Arbeiten beschäftigt, weshalb im ersten Teil der vorliegenden Arbeit dessen Funktion in der Regulation der Chemosensitivität gegenüber dem chlorethylierenden Agenz ACNU genauer untersucht wurde. Es konnte gezeigt werden, dass die FRA 1-Expression durch Behandlung mit ACNU induziert wird. Die Induktion erfolgte über die beiden MAPKs ERK1/2 und p38K. JNK hatte keinen Einfluss auf die Induktion. Durch die Herunterregulation der FRA-1-Expression mit Hilfe von siRNA und eines shRNA exprimierenden Plasmids kam es zu einer signifikanten Sensitivierung gegenüber ACNU. Dabei konnte gezeigt werden, dass die Herunterregulation der FRA-1-Expression in einer verminderten AP 1-Bildung, bedingt durch eine reduzierte Menge an FRA-1 im AP-1-Komplex resultiert. Die Sensitivierung gegenüber ACNU ist weder durch eine Veränderung in der DNA-Reparatur, noch in der Modulation der FAS-Ligand- bzw. FAS-Rezeptor-Expression bedingt. Auch die hier untersuchten BCL 2-Familienmitglieder wiesen keine Unterschiede in der Expression durch Modulation der FRA 1-Expression auf. Allerdings kam es durch die verminderte FRA-1-Expression zu einer Reduktion der Zellzahl in der G2/M-Phase nach Behandlung mit ACNU. Diese ging einher mit einer reduzierten Menge an phosphoryliertem und unphosphoryliertem CHK1, weshalb davon auszugehen ist, dass FRA 1 nach ACNU-Behandlung in Gliomzellen vor der Apoptose schützt, indem es modulierend auf die Zellzykluskontrolle einwirkt.rnIm zweiten Teil dieser Arbeit wurde die Regulation der apoptotischen Antwort nach Behandlung mit ACNU und TMZ genauer beleuchtet, wobei ein spezielles Augen¬merk auf AP 1 und die MAPKs gelegt wurde. Hier konnte gezeigt werden, dass die Apoptose nach Behandlung mit ACNU bzw. TMZ sowohl durch Spaltung von Pro-Caspase 8, als auch Pro-Caspase 9 eingeleitet wird. Dabei akkumulierte in beiden Fällen p53 vermehrt im Zellkern. Eine Inhibierung der transkriptionellen Aktivität von p53 führte nach ACNU-Behandlung zu einer Sensitivierung der Zellen, nach TMZ-Behandlung kam es zu einem leichten Anstieg in der Vitälität. Der FAS-Rezeptor wurde nach ACNU- und nach TMZ-Behandlung aktiviert und auch die DNA-Reparaturproteine DDB2 und XPC wurden in beiden Fällen vermehrt exprimiert. Für die MAPKs JNK und ERK1/2 konnte gezeigt werden, dass diese pro-apoptotisch wirken. Die AP-1-Bildung nach ACNU-Behandlung erfolgte bereits nach 24 h und war von langer Dauer, wohingegen nach TMZ-Behandlung nur eine transiente AP 1-Bildung zu relativ späten Zeitpunkten detektiert werden konnte. Ebenso konnte für das AP-1-Zielgen FAS-Ligand nach ACNU-Behandlung eine relativ schnelle, lang anhaltende Aktivierung detektiert werden, wohingegen nach TMZ-Behandlung zu einem späten Zeitpunkt ein kurzer Anstieg im Signal zu verzeichnen war. In späteren Experimenten konnte gezeigt werden, dass das BCL-2-Familienmitglied BIM eine zentrale Rolle in der Regulation des intrinsischen Apoptosesignalweges nach Behandlung mit ACNU und TMZ spielt. Die hier entstanden Ergebnisse tragen entscheidend zum Verständnis der durch diese beiden Agenzien gesteuerten, apoptotischen Signalwege bei und bieten eine fundierte Grundlage für weitere Untersuchungen.rn

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFalpha could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.

Epidermal caspase-3 cleavage associated with interferon-gamma-expressing lymphocytes in acute atopic dermatitis lesions

Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is believed to play an important role in the pathogenesis of atopic dermatitis (AD), in particular for the formation of spongiosis. To estimate epidermal caspase activation in normal and AD skin under in vivo conditions, we analysed caspase-3 cleavage by immunohistology. In normal skin as well as non-lesional AD skin, we detected caspase-3 cleavage in single cells of the basal layer. In contrast, in acute lesional AD skin, we not only obtained evidence for increased expression of cleaved caspase-3 in keratinocytes of the basal layer but also observed caspase-3 cleavage in one or more layers of the spinous cell layer, in particular in spongiotic areas. Short-term topical treatment of the skin lesions with tacrolimus or pimecrolimus abolished the expression of cleaved caspase-3 in the spinous layer. Moreover, epidermal caspase-3 cleavage correlated with the numbers of dermal interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ lymphocytes in skin lesions of AD patients, supporting the view that IFN-gamma is important for the activation of proapoptotic pathways in keratinocytes. This is also confirmed by the observation of increased Fas expression on keratinocytes in acute AD lesions that was markedly reduced following topical calcineurin inhibitor treatment. These data suggest that caspase-3 cleavage in the spinous layer of the epidermis is a pathologic event contributing to spongiosis formation in AD, whereas cleavage of caspase-3 in basal cells might represent a physiologic mechanism within the process of epidermal renewal.

Human mast cells undergo TRAIL-induced apoptosis

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Markers of endothelial cell activation and immune activation are increased in patients with severe leptospirosis and associated with disease severity

OBJECTIVES Previous studies concluded that haemorrhage is one of the most accurate prognostic factors of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease severity in severe leptospirosis. METHODS Prospective cohort study of severe leptospirosis patients. Plasma levels of sE-selectin and Von Willebrand factor (VWF) were determined. Consequently, an in vitro endothelial cell model was used to assess endothelial activation after exposure to virulent Leptospira. Finally, immune activation, as a potential contributing factor to endothelial cell activation, was determined by soluble IL2-receptor (sIL-2r) and soluble Fas-ligand (sFasL) levels. RESULTS Plasma levels of sE-selectin and VWF strongly increased in patients compared to healthy controls. Furthermore, sE-selectin was significantly elevated (203 ng/ml vs. 157 ng/ml, p < 0.05) in survivors compared to non-survivors. Endothelial cells exposed to virulent Leptospira showed increased VWF expression. E-selectin and ICAM-1 expression did not change. Immunohistochemistry revealed the presence of intracellular Leptospira and qPCR suggested replication. In vivo analysis showed that increased levels of sFasL and sIL-2r were both strongly associated with mortality. Furthermore sIL-2r levels were increased in patients that developed bleeding and significantly correlated to duration of hospital stay. DISCUSSION Markers of endothelial activation and immune activation were associated with disease severity in leptospirosis patients.