Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner

Longitudinal Evaluation of the Hypothalamic-Pituitary-Testicular Function in 8 Boys with Adrenal Hypoplasia Congenita (AHC) Due to NR0B1 Mutations

BACKGROUND:Boys carrying mutations in the NR0B1 gene develop adrenal hypoplasia congenita (AHC) and impaired sexual development due to the combination of hypogonadotropic hypogonadism (HH) and primary defects in spermatogenesis. METHODS:We analysed the evolution of hypothalamic-pituitary-testicular function of 8 boys with AHC due to NR0B1 mutations. Our objective was to characterize and monitor the progressive deterioration of this function. RESULTS:The first symptoms appeared in the neonatal period (n = 5) or between 6 months and 8.7 years (n = 3). Basal plasma adrenocorticotrophic hormone (ACTH) concentrations increased in all boys, whilst cortisol levels decreased in one case. The natremia was equal or below 134 mmol/L and kaliemia was over 5 mmol/L. All had increased plasma renin. In 3 of 4 patients diagnosed in the neonatal period and evaluated during the first year, the basal plasma gonadotropins concentrations, and their response to gonadotropin releasing hormone (GnRH) test (n = 2), and those of testosterone were normal. The plasma inhibin B levels were normal in the first year of life. With the exception of two cases these concentrations decreased to below the normal for age. Anti-Müllerian hormone concentrations were normal for age in all except one case, which had low concentrations before the initiation of testosterone treatment. In 3 of the 8 cases the gene was deleted and the remaining 5 cases carried frameshift mutations that are predicted to introduce a downstream nonsense mutation resulting in a truncated protein. CONCLUSIONS:The decreases in testosterone and inhibin B levels indicated a progressive loss of testicular function in boys carrying NR0B1 mutations. These non-invasive examinations can help to estimate the age of the testicular degradation and cryopreservation of semen may be considered in these cases as investigational procedure with the aim of restoring fertility

Identification of Novel Molecular Targets for Endometrial Cancer Using a Drill-Down LC-MS/MS Approach with iTRAQ

BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma

A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

BACKGROUND: Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels. CONCLUSIONS/SIGNIFICANCE: Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert

The effector T cell response to influenza infection

Influenza virus infection induces a potent initial innate immune response, which serves to limit the extent of viral replication and virus spread. However, efficient (and eventual) viral clearance within the respiratory tract requires the subsequent activation, rapid proliferation, recruitment, and expression of effector activities by the adaptive immune system, consisting of antibody producing B cells and influenza-specific T lymphocytes with diverse functions. The ensuing effector activities of these T lymphocytes ultimately determine (along with antibodies) the capacity of the host to eliminate the viruses and the extent of tissue damage. In this review, we describe this effector T cell response to influenza virus infection. Based on information largely obtained in experimental settings (i.e., murine models), we will illustrate the factors regulating the induction of adaptive immune T cell responses to influenza, the effector activities displayed by these activated T cells, the mechanisms underlying the expression of these effector mechanisms, and the control of the activation/differentiation of these T cells, in situ, in the infected lungs

Granzyme B-induced mitochondrial ROS are required for apoptosis

Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GB-mediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis

Consumo de substâncias psicoativas por adolescentes escolares de Ribeirão Preto, SP (Brasil). I - Prevalência do consumo por sexo, idade e tipo de substância The consumption of psychoactive substances by adolescents in schools in an urban area of Southeastern region of Brazil. I - Prevalence by sex, age and kind of substance

INTRODUÇÃO: A preocupação suscitada quanto ao consumo de substâncias psicoativas pelos adolescentes tem mobilizado grandes esforços em todo o mundo na produção de conhecimento sobre este fenômeno. Decidiu-se estudar as taxas de prevalência de consumo de substâncias psicoativas de uso lícito e ilícito, sua distribuição por idade, sexo e a idade da primeira experiência com essas substâncias, entre adolescentes escolares do Município de Ribeirão Preto, SP, Brasil. MATERIAL DE MÉTODO: Um questionário devidamente adaptado e submetido a um teste de confiabilidade foi auto-aplicado a uma amostra proporcional de 1.025 adolescentes matriculados na oitava série do primeiro grau e primeiro, segundo e terceiro anos do segundo grau, das escolas públicas e privadas do município estudado. O questionário continha questões sobre o uso de dez classes de substâncias psicoativas, questões demográficas e informações de validação, além de questões de percepção e comportamento intrínseco ao consumo de drogas. RESULTADOS: Da amostra 88,9% consumiram bebidas alcoólicas alguma vez na vida; 37,7% utilizaram o tabaco; 31,1% os solventes; 10,5% os medicamentos; 6,8% a maconha; 2,7% a cocaína; 1,6% os alucinógenos e 0,3% consumiu alguma substância a base de opiácios. As taxas de consumo cresceram com a idade, para todas as substâncias; no entanto, o uso de tabaco e de substâncias ilícitas mostrou uma desaceleração nos anos que compreendem o final da adolescência. Verificou-se que os meninos consumiram mais do que as meninas, exceto para os medicamentos, com as meninas consumindo barbitúricos, anfetaminas e tranqüilizantes em proporções semelhantes ou maiores que os meninos. A idade da primeira experiência mostrou que o acesso às substâncias psicoativas ocorreu em idades bastante precoces. CONCLUSÕES: As substâncias psicoativas, sejam lícitas ou ilícitas, são freqüentemente experimentadas na adolescência, tanto pelos meninos como pelas meninas, muitas vezes em idades bem precoces.<br>INTRODUCTION: Concern over the consumption of psychoactive substances by teenagers has given rise to a great wordwide effort to produce information about this phenomenon. This study set out to investigate the prevalence of consumption of legal and illegal psychoactive substances, its distribution by age, sex and age at first experience of them, among teenage pupils in county, Ribeirão Preto, SP, Southeastern Brazil. MATERIAL AND METHOD: A self-applicable questionnaire duly adapted and submitted to a reliability test was applied to a proportional sample of 1,025 teenagers enrolled in 8th, 9th, 10th and 11th grads at public and private city schools. The questionnaire contained questions about the use of ten classes of psychoactive substances, demographic questions and validation information, as well as questions about the perception and intrinsic behavior related to drug consumption. RESULTS: The sample of 88.9% had consumed alcoholic beverages sometime in their lives, 37.7% had used tobacco, 31.1% solvents, 10.5% medicines, 6.8% marihuana, 2.7% cocaine, 1.6% hallucinogens, and 0.3% of the sample had consumed some opiate substance. The rates of consumption increased with age for all substances; however, the use of tobacco and of illegal substances was less intense during the later years of adolescence. As to sex distribution, boys consumed more than girls, except for medicines, with girls consuming barbiturates, amphetamines and tranquilizers in proportions similar to or higher than those observed among boys. Age at first experience showed that access to psychoactive substances occurred at very early ages. CONCLUSIONS: Experimenting with psychoactive substances, whether legal or illegal, is a frequent phenomenon during adolescence, both among boys and girls, often at very early ages