Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

The response of soil organic carbon of a rich fen peatland in interior Alaska to projected climate change

It is important to understand the fate of carbon in boreal peatland soils in response to climate change because a substantial change in release of this carbon as CO₂ and CH₄ could influence the climate system. The goal of this research was to synthesize the results of a field water table manipulation experiment conducted in a boreal rich fen into a process‐based model to understand how soil organic carbon (SOC) of the rich fen might respond to projected climate change. This model, the peatland version of the dynamic organic soil Terrestrial Ecosystem Model (peatland DOS‐TEM), was calibrated with data collected during 2005–2011 from the control treatment of a boreal rich fen in the Alaska Peatland Experiment (APEX). The performance of the model was validated with the experimental data measured from the raised and lowered water‐table treatments of APEX during the same period. The model was then applied to simulate future SOC dynamics of the rich fen control site under various CO₂ emission scenarios. The results across these emissions scenarios suggest that the rate of SOC sequestration in the rich fen will increase between year 2012 and 2061 because the effects of warming increase heterotrophic respiration less than they increase carbon inputs via production. However, after 2061, the rate of SOC sequestration will be weakened and, as a result, the rich fen will likely become a carbon source to the atmosphere between 2062 and 2099. During this period, the effects of projected warming increase respiration so that it is greater than carbon inputs via production. Although changes in precipitation alone had relatively little effect on the dynamics of SOC, changes in precipitation did interact with warming to influence SOC dynamics for some climate scenarios.     

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.     

Using a Human Challenge Model of Infection to Measure Vaccine Efficacy: A Randomised,Controlled Trial Comparing the Typhoid Vaccines M01ZH09 with Placebo and Ty21a

BackgroundTyphoid persists as a major cause of global morbidity. While several licensed vaccines to prevent typhoid are available, they are of only moderate efficacy and unsuitable for use in children less than two years of age. Development of new efficacious vaccines is complicated by the human host-restriction of Salmonella enterica serovar Typhi (S. Typhi) and lack of clear correlates of protection. In this study, we aimed to evaluate the protective efficacy of a single dose of the oral vaccine candidate, M01ZH09, in susceptible volunteers by direct typhoid challenge.ConclusionsDespite successfully demonstrating the use of a human challenge study to directly evaluate vaccine efficacy, a single-dose M01ZH09 failed to demonstrate significant protection after challenge with virulent Salmonella Typhi in this model. Anti-Vi antibody detected prior to vaccination played a major role in outcome after challenge.

Trial registration

ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT01405521","term_id":"NCT01405521"}}NCT01405521) and EudraCT (number 2011-000381-35).     

Identification and quantitation of unique fatty acid oxidation products in human atherosclerotic plaque using high-performance liquid chromatography

Oxidation of lipoproteins, particularly low-density lipoprotein, is thought to play a major role in the development of atherosclerosis. We set out to identify and quantitate the major fatty acid oxidation products in human atherosclerotic plaque obtained from individuals undergoing carotid endarterectomy. Oxidized lipids were extracted from plaque homogenate under conditions to prevent artifactual oxidation. Identification and quantitation was performed using HPLC and GC-MS. High levels of hydroxyoctadecanoic acids (0.51 +/- 0.17 ng/microg of linoleic acid), 15-hydroxyeicosatetranoic acid (HETE) (0.66 +/- 0.24 ng/microg of arachidonic acid), and 11-HETE (0.84 +/- 0.24 ng/microg of arachidonic acid) were detected in all atherosclerotic plaques (n = 10). Low levels of 9-oxo-octadecanoic acid (oxoODE) (0.04 +/- 0.01 ng/microg of linoleic acid), were present in all samples, while 13-oxoODE (0.01 +/- 0.008 ng/microg of linoleic acid) was present in only 4 of the 10 plaque samples. Of interest was the identification of two previously unidentified compounds in atherosclerotic plaque, 11-oxo-eicosatetranoic acid in 9 of the 10 samples and 5,6-dihydroxyeicosatetranoic acid in 3 samples. Chiral analysis revealed that all the major compounds identified in this study are of a nonenzymatic origin. This study is the first to provide a convenient HPLC method to quantify all the products of both linoleic acid and arachidonic acid oxidation in human atherosclerotic plaque. The quantitation of lipid peroxidation products in plaque may be important given the potential biological activity of these compounds and their possible relationship to plaque pathogenesis and instability.     

Analysis of telomere length in Dolly, a sheep derived by nuclear transfer

We have used a (TTAGGG) oligonucleotide probe to demonstrate that ovine telomeres are composed of (TTAGGG) repeat arrays and to compare the terminal restriction fragment lengths of sheep derived by natural mating and nuclear transfer. Here we show that ovine somatic telomeres decrease in length with age, and that Dolly, derived by the transfer of 6-year-old adult somatic nucleus, exhibits diminished terminal restriction fragment lengths. The decrease is consistent with the age of the donor tissue and telomere erosion during in vitro culture. Nuclear transfer does not restore telomere lengths. Dolly otherwise appears physiologically and phenotypically normal for her breed and age. We further report on apparent telomere lengthening in sheep, occurring during the first year in naturally derived lambs.     

Cost-intake information used in foraging

An aspect of the round dance, called RATE, was used as quantification of 15 honey bees' (Apis mellifera) perceptions of caloric costs and intakes experienced while foraging at artificial flowers. Caloric intake per floral visit (CALGAIN) was manipulated by varying sucrose concentration. Both flight and handling costs per floral visit (CALCOST) were manipulated. Multiple linear regression analysis was used to quantify the relationship between the dependent variable, RATE, and the two energy variables. There was significant variation among bees in dance behaviour. However, the absolute magnitude of the CLACOST coefficients tended to be larger than CALGAIN coefficients; bees may weight perceptions of costs in relation to intakes. Honey bees were presented with a series of binary choices between yellow or blue tubular artificial flowers in order to assess how cost-intake information is used to make choices among flowers. In eight experiments the 2 flowers had different combinations of volume of sucrose (intake) and tube depth (handling cost). Results are consistent with an assessment of rate of net caloric intake, although they are likely consistent with other possible assessments.     

Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi Challenge Model

Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes significant morbidity and mortality worldwide. Currently available vaccines are moderately efficacious, and identification of immunological responses associated with protection or disease will facilitate the development of improved vaccines. We investigated S. Typhi-specific modulation of activation and homing potential of circulating regulatory T cells (T_reg) by flow and mass cytometry using specimens obtained from a human challenge study. Peripheral blood mononuclear cells were obtained from volunteers pre- and at multiple time-points post-challenge with wild-type S. Typhi. We identified differing patterns of S. Typhi-specific modulation of the homing potential of circulating T_reg between volunteers diagnosed with typhoid (TD) and those who were not (No TD). TD volunteers demonstrated up-regulation of the gut homing molecule integrin α4ß7 pre-challenge, followed by a significant down-regulation post-challenge consistent with T_reg homing to the gut. Additionally, S. Typhi-specific T_reg from TD volunteers exhibited up-regulation of activation molecules post-challenge (e.g., HLA-DR, LFA-1). We further demonstrate that depletion of T_reg results in increased S. Typhi-specific cytokine production by CD8+ T_EM in vitro. These results suggest that the tissue distribution of activated T_reg, their characteristics and activation status may play a pivotal role in typhoid fever, possibly through suppression of S. Typhi-specific effector T cell responses. These studies provide important novel insights into the regulation of immune responses that are likely to be critical in protection against typhoid and other enteric infectious diseases.