Performance of a household tuberculosis exposure survey among children in a Latin American setting.

OBJECTIVE: To evaluate the performance of a survey that quantifies the intensity of household tuberculosis (TB) exposure among children.METHODS: Children aged 0-14 years in Lima, Peru, with ≥1 signs and/or symptoms of TB and a history of contact with an adult TB patient were included. The 10-question survey was administered to caregivers and addressed sleep proximity, frequency of exposure, and infectiousness of the contact. Infection status was determined using tuberculin skin tests (TSTs). The exposure scale was evaluated for association with TST positivity using mixed-effects regression analyses.RESULTS: The exposure score was significantly associated with TST positivity (age-adjusted odds ratio [aOR] 1.14, 95%CI 1.02-1.28). We observed a stronger association with TST positivity in children aged ≤5 years; (aOR 1.23, 95%CI 1.07-1.41) and no association in children 6-14 years of age (aOR 0.99, 95%CI 0.82-1.20).CONCLUSION: This survey was easy to use and modestly successful in predicting TST positivity in children aged ≤5 years. It may be a useful resource for clinicians for diagnosing TB in children, and for national TB programs aiming to scale up preventive therapy initiatives.

Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor.

BACKGROUND: Natural killer (NK) cells are important anti-tumor cells of our innate immune system. Their anti-cancer activity is mediated through interaction of a wide array of activating and inhibitory receptors with their ligands on tumor cells. After activation, NK cells also secrete a variety of pro-inflammatory molecules that contribute to the final immune response by modulating other innate and adaptive immune cells. In this regard, external proteins from NK cell secretome and the mechanisms by which they mediate these responses are poorly defined. METHODS: TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) combined with proteomic was undertaken to identify early materials transferred between cord blood-derived NK cells (CB-NK) and multiple myeloma (MM) cells. Further in vitro and in vivo studies with knock-down of histones and CD138, overexpression of histones and addition of exogenous histones were undertaken to confirm TRANS-SILAC results and to determine functional roles of this material transferred. RESULTS: We describe a novel mechanism by which histones are actively released by NK cells early after contact with MM cells. We show that extracellular histones bind to the heparan sulfate proteoglycan CD138 on the surface of MM cells to promote the creation of immune-tumor cell clusters bringing immune and MM cells into close proximity, and thus facilitating not only NK but also T lymphocyte anti-MM activity. CONCLUSION: This study demonstrates a novel immunoregulatory role of NK cells against MM cells mediated by histones, and an additional role of NK cells modulating T lymphocytes activity that will open up new avenues to design future immunotherapy clinical strategies.

Cytochrome c is released in a single step during apoptosis.

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

Interferon-gamma treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program.

In this report, we have assessed the role of IFN-gamma as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-gamma significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-gamma treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-gamma. Furthermore, IFN-gamma sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-gamma, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.

Effects of extremely low frequency electromagnetic fields on in-vitro cellular cultures HeLa and CHO.

This paper presents the cellular proliferation effects of the exposure to extremely low frequency electromagnetic fields (ELF-EMF) on in-vitro cellular cultures HeLa and CHO. Through the magnetic stimulation system (MSS) the cells were exposed to magnetic fields with sinusoidal waveform at 50 Hz; initially for 40 minutes at intensities of 0.4 mT, 1.4 mT, 2.13 mT, 2.49 mT and 2.53 mT in parallel and perpendicular directions to the culture plates. Subsequently, the repetitive electromagnetic field (rEMF) was applied to 2.49 mT in parallel direction (for 40 minutes every twelve hours during 4 days) with which the highest cellular proliferation rate was obtained at 66.6 %. The results show a greater effect on proliferation in radiated cell lines, particularly in the application of rEMF a greater effect of ELF-EMF was observed in the proliferation rate of HeLa cells than in CHO cells, in contrast to the respective control cells. These results supported by other studies serve as a reference in the search for alternatives for the treatment of cervical cancer and the maintenance and preservation of cell lines.

The differential sensitivity of Bc1-2-overexpressing human breast tumor cells to TRAIL or doxorubicin-induced apoptosis is dependent on Bc1-2 protein levels.

Bc1-2 protein is a potent anti-apoptotic protein that inhibits a mitochondria-operated pathway of apoptosis in many cells. DNA damaging agents and death receptor ligands can activate this mitochondrial apoptotic mechanism. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been suggested to escape from the inhibitory action of Bc1-2 protein. We show that in human breast tumor MCF-7 cells, TRAIL induced a mitochondrial pathway of apoptosis that involved cytochrome c release from mitochondria and activation of caspase 9. The DNA damaging drug doxorubicin also activated this mitochondria-regulated mechanism of apoptosis, which was inhibited in Bc1-2-overexpressing cells. We also demonstrate that in MCF-7 cells Bc1-2 might confer resistance to TRAIL-induced apoptosis, depending on the expression levels of the anti-apoptotic protein. These results indicate that enhanced expression of Bc1-2 in tumor cells can render these cells less sensitive not only to chemotherapeutic drugs but also to TRAIL.

Bid acts on the permeability transition pore complex to induce apoptosis.

Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transition pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking.

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid-protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

Effects of the ionophore anticoccidial semduramicin on broiler breeders.

Three experiments were conducted to assess the effects on broiler breeders of contamination of feed with the ionophore anticoccidial semduramicin. In Experiment 1, individually caged females received 0, 12.5, or 25 mg/kg diet for 3 wk from 48 to 50 wk of age. In Experiment 2, males and females in floor pens received 0, 12.5, or 25 mg/kg diet for 3 wk from 63 to 65 wk of age. In Experiment 3, individually caged males and females received 0, 3, 6, or 25 mg/kg diet for 1 wk at 31 wk of age and were mated by artificial insemination. There was a dose-related decrease in cumulative egg production and percentage shell in Experiment 1 after more than 1 wk exposure, but these effects were not observed in the other experiments. There was a decrease in cumulative fertile hatchability and a dose-related decrease after 3 wk exposure due to an increase in early embryonic mortality in Experiment 2, but these changes were not observed during the 1-wk exposure in Experiment 3. The data show that adverse effects of semduramicin require greater than 1 wk of exposure to be evident.

Bebidas energéticas y estudiantes universitarios en España. Usos, efectos y asociaciones / Energy drinks and university students in Spain. Uses, effects and associations

INTRODUCCIÓN: Las bebidas energéticas (BE) son bebidas carbonatadas creadas y comercializadas con la intención de incrementar el rendimiento físico y mental. Sus ingredientes principales son la glucosa, cafeína, taurina, glucoronolactona y vitaminas del grupo B. OBJETIVOS: Conocer la frecuencia de consumo de BE en estudiantes universitarios, la finalidad, así como los factores asociados a su uso, su relación con el consumo de otras sustancias y los efectos experimentados. MATERIAL Y MÉTODOS: Estudio de corte transversal realizado a través de una encuesta online por medio de la plataforma "Formularios" de Google DocsR, lanzada entre marzo y agosto de 2014 dirigido a estudiantes universitarios de toda España. Los resultados se analizaron mediante el paquete estadístico SPSSR. RESULTADOS: De los 633 encuestados 221 fueron hombres (34.9 %) y 412 mujeres (65.1 %).384 (61 %) han probado las BE. El consumo es mayor en hombres ,70 %, frente a un 55% en mujeres. Dentro de sus efectos reportados están: el incremento rendimiento académico (23.7 %) taquicardia (34.9 %), el insomnio (33.6 %) y nerviosismo (45.1 %). Hemos encontrado asociación entre su consumo con: psicofármacos; de drogas en general; alcohol; tabaco y marihuana (OR=1.43; 1.28; 1.5; 1.25; 1.28, respectivamente). En época de exámenes observamos una asociación entre consumo de BE y el cambio de dieta (OR= 1.65). Finalmente, existe una asociación positiva ente la toma de BE con intención de mejorar el rendimiento académico y la sensación de obtenerlo (OR=2.72). CONCLUSIONES: Existe un alto consumo de BE entre los estudiantes universitarios, predominante en varones, asociado a la época de exámenes y su consumo junto a bebidas alcohólicas. Se encuentra una asociación con la toma separada de alcohol, tabaco, marihuana y psicofármacos. Es por ello que se cree conveniente llevar a cabo estudios que aborden posibles asociaciones de su consumo con alteraciones en la dieta

INTRODUCTION: Energy drinks are carbonated drinks which purpose is to increase physical and mental performance. Its main ingredients are glucose, caffeine, taurine, glucoronolactone and vitamine B. MAIN OBJECTIVE: We aim to asses the frequency of the use of energy drinks and its purpose, among a population of university students. We aim also to correlate it with the use of other substances and their effects. MATERIAL AND METHODS: Cross- sectional study designed throug an on line questionnaire launched in the Google DocsR platform, and answered by spanish university students between march and august of 2014. Statistical analysis was performed with statistical software SPSSR. RESULTS: Out of 633 participants 221 were male (34,9%) and 412 female (65,1%).384 (61%) had tried energetic drinks. Its use is higher in male (70%) than in female (55%). Between its effects we found the increase in academic performance (23,7%), tachycardia (34,9%), insomnia (33,6%), and anxiety (45,1%). We found an association between the intake of energy drinks and the use of psychoactive drugs, abuse drugs, alcohol, tobacco and marijuana (OR=1.43; 1.28; 1.5; 1.25; 1.28). During exams period we found an association between intake of energy drinks and change in feeding habits (OR= 1.65). Finally, there is an association between the intake of energy drinks with the purpose of increasing academic performance and the feeling of having achieved it (OR=2.72). CONCLUSIONS: There is a high prevalence of energy drinks intake between university students, specially in male, associated to exams period and in combination with alcoholic drinks. There is an association with the use of alcohol, tobacco, marijuana and psychoactive drugs. We suggest it woud be convenient to design tryals to find out the association between its use and change in diet habits

Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein.

Apolipoproteins of the L family are lipid-binding proteins whose function is largely unknown. Apolipoprotein L1 and apolipoprotein L6 have been recently described as novel pro-death BH3-only proteins that are also capable of regulating autophagy. In an in-silico screening to discover novel putative BH3-only proteins, we identified yet another member of the apolipoprotein L family, apolipoprotein L2 (ApoL2), as a BH3 motif-containing protein. ApoL2 has been suggested to behave as a BH3-only protein and mediate cell death induced by interferon-gamma or viral infection. As previously described, we observed that ApoL2 protein was induced by interferon-gamma. However, knocking down its expression in HeLa cells did not regulate cell death induced by interferon-gamma. Overexpression of ApoL2 did not induce cell death on its own. ApoL2 did not sensitize or protect cells from overexpression of the BH3-only proteins Bmf or Noxa. Furthermore, siRNA against ApoL2 did not alter sensitivity to a variety of death stimuli. We could, however, detect a weak interaction between ApoL2 and Bcl-2 by immunoprecipitation of the former, suggesting a role of ApoL2 in a Bcl-2-regulated process like autophagy. However, in contrast to what has been described about its homologs ApoL1 and ApoL6, ApoL2 did not regulate autophagy. Thus, the role, if any, of ApoL2 in cell death remains to be clarified.

Analysis of BH3-only proteins upregulated in response to oxygen/glucose deprivation in cortical neurons identifies Bmf but not Noxa as potential mediator of neuronal injury.

Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

Cancer metabolism: current perspectives and future directions.

Cellular metabolism influences life and death decisions. An emerging theme in cancer biology is that metabolic regulation is intricately linked to cancer progression. In part, this is due to the fact that proliferation is tightly regulated by availability of nutrients. Mitogenic signals promote nutrient uptake and synthesis of DNA, RNA, proteins and lipids. Therefore, it seems straight-forward that oncogenes, that often promote proliferation, also promote metabolic changes. In this review we summarize our current understanding of how 'metabolic transformation' is linked to oncogenic transformation, and why inhibition of metabolism may prove a cancer's 'Achilles' heel'. On one hand, mutation of metabolic enzymes and metabolic stress sensors confers synthetic lethality with inhibitors of metabolism. On the other hand, hyperactivation of oncogenic pathways makes tumors more susceptible to metabolic inhibition. Conversely, an adequate nutrient supply and active metabolism regulates Bcl-2 family proteins and inhibits susceptibility to apoptosis. Here, we provide an overview of the metabolic pathways that represent anti-cancer targets and the cell death pathways engaged by metabolic inhibitors. Additionally, we will detail the similarities between metabolism of cancer cells and metabolism of proliferating cells.

Sugar-free approaches to cancer cell killing.

Tumors show an increased rate of glucose uptake and utilization. For this reason, glucose analogs are used to visualize tumors by the positron emission tomography technique, and inhibitors of glycolytic metabolism are being tested in clinical trials. Upregulation of glycolysis confers several advantages to tumor cells: it promotes tumor growth and has also been shown to interfere with cell death at multiple levels. Enforcement of glycolysis inhibits apoptosis induced by cytokine deprivation. Conversely, antiglycolytic agents enhance cell death induced by radio- and chemotherapy. Synergistic effects are likely due to regulation of the apoptotic machinery, as glucose regulates activation and levels of proapoptotic BH3-only proteins such as Bim, Bad, Puma and Noxa, as well as the antiapoptotic Bcl-2 family of proteins. Moreover, inhibition of glucose metabolism sensitizes cells to death ligands. Glucose deprivation and antiglycolytic drugs induce tumor cell death, which can proceed through necrosis or through mitochondrial or caspase-8-mediated apoptosis. We will discuss how oncogenic pathways involved in metabolic stress signaling, such as p53, AMPK (adenosine monophosphate-activated protein kinase) and Akt/mTOR (mammalian target of rapamycin), influence sensitivity to inhibition of glucose metabolism. Finally, we will analyze the rationale for the use of antiglycolytic inhibitors in the clinic, either as single agents or as a part of combination therapies.

Prácticas de Salud Ocupacional y niveles de biomarcadores séricos en aplicadores de plaguicidas de cultivos de arroz en Natagaima-Tolima, Colombia / Occupational health practice and levels of serum biomarkers in pesticide applicators rice crops in Natagaima-Tolima, Colombia

Los pequeños agricultores de arroz del sector informal en Colombia son una población vulnerable a la intoxicación aguda y crónica por plaguicidas, especialmente porque no han sido incluidos en programas nacionales de capacitación, vigilancia y control de intoxicaciones. El objetivo de este estudio fue identificar mediante una encuesta el estado de cumplimiento de las prácticas de salud ocupacional y disposición adecuada de residuos de plaguicidas en un grupo de agricultores del municipio de Natagaima en Colombia y evaluar el nivel de biomarcadores séricos de efecto por exposición a plaguicidas en esta población. Los resultados muestran un panorama en donde prevalece una aparente percepción del riesgo por el uso de plaguicidas, pero hay una gran carencia de prácticas de salud ocupacional en la población que los manipula. El análisis de los biomarcadores séricos permitió detectar un aumento significativo en los niveles de aspartato amino transferasa, creatinina y ácido úrico y descenso de los niveles de colinesterasa sérica demostrando posibles alteraciones subclínicas de la función renal y hepática en la población estudiada (AU)

Small farmers in rice informal sector in Colombia are vulnerable to acute and chronic pesticide poisoning population, especially because they have not been included in national training programs, monitoring and poison control. The aim of this study was to identify through a survey of the state of compliance with occupational health practices and proper disposal of pesticide residues in a group of farmers in the municipality of Natagaima in Colombia and assess the level of serum biomarkers of effect exposure pesticides in this population. The results show a scenario in which prevails an apparent perception of risk by the use of pesticides, but there is a lack of practical occupational health in the population that manipulates

Glycolysis inhibition sensitizes tumor cells to death receptors-induced apoptosis by AMP kinase activation leading to Mcl-1 block in translation.

Most cancer cells exhibit increased glycolysis for generation of their energy supply. This specificity could be used to preferentially kill these cells. In this study, we identified the signaling pathway initiated by glycolysis inhibition that results in sensitization to death receptor (DR)-induced apoptosis. We showed, in several human cancer cell lines (such as Jurkat, HeLa, U937), that glucose removal or the use of nonmetabolizable form of glucose (2-deoxyglucose) dramatically enhances apoptosis induced by Fas or by tumor necrosis factor-related apoptosis-inducing ligand. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. We established the fact that AMPK is activated upon glycolysis block resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease, but no other Bcl-2 anti-apoptotic members. Interestingly, we determined that, upon glycolysis inhibition, the AMPK-mTOR pathway controlled Mcl-1 levels neither through transcriptional nor through posttranslational mechanism but rather by controlling its translation. Therefore, our results show a novel mechanism for the sensitization to DR-induced apoptosis linking glucose metabolism to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of DR ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

Glucose deprivation induces an atypical form of apoptosis mediated by caspase-8 in Bax-, Bak-deficient cells.

Apoptosis induced by most stimuli proceeds through the mitochondrial pathway. One such stimulus is nutrient deprivation. In this study we studied death induced by glucose deprivation in cells deficient in Bax and Bak. These cells cannot undergo mitochondrial outer membrane permeabilization (MOMP) during apoptosis, but they undergo necrosis when treated with MOMP-dependent apoptotic stimuli. We find in these cells that glucose deprivation, rather than inducing necrosis, triggered apoptosis. Cell death required caspase activation as inhibition of caspases with peptidic inhibitors prevented death. Glucose deprivation-induced death displayed many hallmarks of apoptosis, such as caspase cleavage and activity, phosphatidyl-serine exposure and cleavage of caspase substrates. Neither overexpression of Bcl-xL nor knockdown of caspase-9 prevented death. However, transient or stable knockdown of caspase-8 or overexpression of CrmA inhibited apoptosis. Cell death was not inhibited by preventing death receptor-ligand interactions, by overexpression of c-FLIP or by knockdown of RIPK1. Glucose deprivation induced apoptosis in the human tumor cell line HeLa, which was prevented by knockdown of caspase-8. Thus, we have found that glucose deprivation can induce a death receptor-independent, caspase-8-driven apoptosis, which is engaged to kill cells that cannot undergo MOMP.