Dynamic changes of circulating miRNAs induced by the Ebola virus vaccine VSV-EBOV.

VSV-EBOV is a replication-competent Ebola virus (EBOV) vaccine, which was tested in clinical trials as response to the Ebola virus disease (EVD) outbreak 2013-2016. It is the most advanced EBOV candidate currently in the licensure process. The experimental vaccine was again administered as response to outbreaks in the Democratic Republic of Congo. However, underlying molecular mechanisms that convey protection remain incompletely understood. MicroRNAs (miRNAs) are known key regulators that influence gene expression on a post-transcriptional level. The miRNA-mediated control has emerged as a critical regulatory principle in the immune system, which strongly influences the balance of innate and adaptive immune responses by modulation of signaling pathways critical for differentiation of immune cells. We investigated expression levels of circulating miRNAs (c-miRNAs) in plasma from healthy vaccinees, as they may reflect cellular dynamics following VSV-EBOV immunization and additionally may serve as potential biomarkers for vaccine efficacy. As part of the WHO-led VEBCON consortium, we investigated safety and immunogenicity of VSV-EBOV in a phase I trial. A comprehensive analysis of expression levels on c-miRNAs from plasma samples following VSV-EBOV immunization (day 0, 1, 3 post vaccination) was conducted using RT-qPCR assays. Potential biological relevance was assessed using in silico analyses. Additionally, we correlated dynamics of miRNA expressions with our previously reported data on vaccine-induced antibody and cytokine responses and finally evaluated the prognostic power by generating ROC curves. We identified four promising miRNAs (hsa-miR-146a, hsa-miR-126, hsa-miR-199a, hsa-miR-484), showing a strong association with adaptive immune responses, exhibited favourable prognostic performance and are implicated in immunology-related functions. Our results provide evidence that miRNAs may serve as useful biomarkers for prediction of vaccine-induced immunogenicity. Furthermore, our unique data set provides insight into molecular mechanisms that underlie VSV-EBOV-mediated protective immune responses, which may help to decipher VSV-EBOV immune signature and accelerate strategic vaccine design or personalized approaches.

Violent societies: an application of orbital decomposition to the problem of human violence.

This study uses orbital decomposition to analyze the patterns of how governments lose their monopolies on violence, therefore allowing those societies to descend into violent states from which it is difficult to recover. The nonlinear progression by which the governing body loses its monopoly is based on the work of criminologist Lonnie Athens and applied from the individual to the societal scale. Four different kinds of societies are considered: Those where the governing body is both unwilling and unable to assert its monopoly on violence (former Yugoslavia); where it is unwilling (Peru); where it is unable (South Africa); and a smaller pocket of violent society within a larger, more stable one (Gujarat). In each instance, orbital decomposition turns up insights not apparent in the qualitative data or through linear statistical analysis, both about the nature of the descent into violence and about the progression itself.

Insights into the biochemical mechanism of maleic acid-induced Fanconi syndrome.

Maleic acid administration is known to produce the Fanconi syndrome, although the biochemical mechanism is incompletely understood. In this study the effect of a single injection of maleic acid (50 mg/kg body wt, i.v.) on the rat renal ATPases was examined. Maleic acid rapidly caused bicarbonaturia, natriuresis, and kaliuresis. When nephron segments were microdissected, there was an 81 +/- 2% reduction in proximal convoluted tubule (PCT) Na-K-ATPase activity (P < 0.005) and a 48 +/- 4% reduction in PCT H-ATPase activity (P < 0.01). Enzyme activity (Na-K-ATPase, H-ATPase, H-K-ATPase) in the medullary thick ascending limb of Henle's loop and distal nephron segments was normal. In vitro, maleic acid (1 and 10 mM) inhibited Na-K-ATPase in PCT, but it had no effect on H-ATPase in PCT. Prior phosphate infusion to maleic acid-treated rats attenuated urinary bicarbonate wastage by 50% (P < 0.05); activity of proximal tubule Na-K-ATPase and H-ATPase activities were partially protected as compared to the animals given maleic acid alone (P < 0.05). Renal cortical ATP levels were not altered at the concentration of maleic acid used in this study (that is, 50 mg/kg body wt), but higher doses of maleic acid (that is, 500 and 1000 mg/kg body wt) caused ATP levels to fall. Maleic acid did not affect cortical medullary total phosphate concentration, however, P32 turnover (1 and 24 hr) was altered by prior phosphate infusion. A protective effect of prior phosphate loading on the membrane bound Pi pool (insoluble) was seen while the cytosolic Pi pool (soluble) was not different from control. Thus, maleic acid-induced "Fanconi" syndrome likely results from both direct inhibition of proximal tubule Na-K-ATPase activity and membrane-bound phosphorus depletion. The former mechanism would reduce activity of the sodium-dependent transporters (that is, Na/H antiporter), while the latter would inhibit the electrogenic proton pump (H-ATPase). The combination of reduced proximal tubule Na-H exchange and H-ATPase activities would markedly inhibit bicarbonate reabsorption and result in the metabolic acidosis universally seen in the Fanconi syndrome.

Effects of pH on calcium transport in turtle bladder.

This study was designed to examine the effect of apical and basolateral (ie, mucosal and serosal) pH on calcium (Ca) transport in turtle bladder, a nonmammalian analog of the distal nephron. Unidirectional Ca45 fluxes were measured when serosal pH was 6.4, 7.4, or 8.4 (mucosal pH, 7.4) in the presence and absence of ouabain. When serosal pH was 8.4, M-->S Ca45 flux increased significantly, and when it was 6.4, M-->S Ca45 flux decreased markedly. Changes in serosal pH did not affect the S-->M Ca45 flux. When 5 x 10(-4) mol/L ouabain was added to inhibit sodium transport, M-->S Ca45 flux, at pH 7.4, was 221.6 +/- 27.4 pmol/mg/h (n = 10), and low pH again inhibited this flux (approximately 50%). Lowering mucosal pH (with serosal pH 7.4) also decreased M-->S Ca45 flux. In stripped bladders, Ca45 uptake increased linearly as medium pH was increased from 4.4 to 8.4. Total tissue Ca concentration did not change when serosal pH was varied, except at the extreme of pH 4.4, where tissue Ca decreased. By contrast, when apical pH was 6.4, tissue Ca rose substantially (approximately 1.5-fold). these results demonstrate that extracellular pH directly affects Ca homeostasis in the turtle bladder. Lowering the pH of either the serosal or mucosal medium directly inhibits apical Ca permeability. This change in Ca permeability is seen in the presence of ouabain. By contrast, alkalization of the serosal medium enhances apical permeability, but this effect is, in some manner, related to sodium transport.(ABSTRACT TRUNCATED AT 250 WORDS)

H-K-ATPase in distal renal tubular acidosis: urinary tract obstruction, lithium, and amiloride.

In previous studies we suggested that urinary tract obstruction and chronic administration of lithium or amiloride were models of "voltage-dependent" distal renal tubular acidosis (DRTA). Subsequently, differences among these three models suggested that the pathogenesis was far more complex than we originally proposed. A recent study showed that H-adenosinetriphosphatase (H-ATPase) activity was decreased in all three experimental models. In the current experiments we examined the effect of 24-h unilateral ureteral obstruction (UUO) and chronic administration of amiloride and lithium on collecting tubule H-K-ATPase, the other renal H-ATPase enzyme. In the obstructed kidney, cortical collecting tubule (CCT) H-K-ATPase activity was enhanced by 73 +/- 10.0%, whereas the enzyme activity in medullary collecting tubule (MCT) was decreased by 67 +/- 5.4%. In the normal contralateral kidney, activities of H-ATPase, H-K-ATPase, and Na-K-ATPase were increased by approximately 30% in both CCT and MCT. Following amiloride (3 mg.kg-1.day-1 x 3 days ip), rats had normal acid-base status, slight hyperkalemia, and markedly elevated plasma aldosterone levels. Both CCT and MCT H-K-ATPase activities in amiloride-treated rats were unchanged. After LiCl (4 meq.kg-1.day-1 x 3 days ip), rats developed mild metabolic acidosis and had normokalemia and normal aldosterone status. CCT H-K-ATPase activity in lithium-treated rats was decreased by 64 +/- 8.8%, whereas the enzyme activity in MCT remained unchanged. Lithium in vitro (30 meq/l) inhibited CCT, but not MCT, H-K-ATPase activity, whereas amiloride had no effect on the enzyme activity. (ABSTRACT TRUNCATED AT 250 WORDS)

Vanadate causes hypokalemic distal renal tubular acidosis.

Considerable evidence supports the presence of an H(+)-K(+)-ATPase along the mammalian nephron. Inhibition of this enzyme might be expected to reduce acid excretion while increasing potassium excretion, thus causing hypokalemic distal renal tubular acidosis (RTA). In this study we administered vanadate at a dose of 5 mg/kg ip for 10 days to rats. These animals developed hypokalemic distal RTA with a blood pH of 7.22 +/- 0.01, a plasma bicarbonate of 15.2 +/- 0.6 meq/l, and a plasma potassium of 3.28 +/- 0.06 meq/l. The vanadate-treated animals had a urine pH of 6.70 +/- 0.09, a value significantly higher than NH4Cl-treated animals with the same degree of acidemia (urine pH = 5.25 +/- 0.04). When cortical collecting tubules (CCT) from these animals were microdissected and H(+)-K(+)-ATPase was measured, it was decreased by approximately 75% (P less than 0.001); but H(+)-ATPase was no different from control. In medullary collecting tubule, H(+)-K(+)-ATPase was also decreased but less than in CCT. Muscle potassium concentration in the vanadate-treated animals was significantly lower than in controls. These results demonstrate that vanadate causes hypokalemic distal RTA in association with inhibition of collecting tubule H(+)-K(+)-ATPase activity.

Characterization of the N-ethylmaleimide-sensitive ATPase in rat cortical and medullary collecting tubule.

Hydrogen ion secretion in the kidney is thought to be mediated in part by an N-ethylmaleimide (NEM)-sensitive proton-translocating adenosine triphosphatase (ATPase). This enzyme has been found throughout the nephron, but it has not been completely characterized enzymatically in the rat collecting duct. In the present study we characterized the NEM-sensitive ATPase from microdissected cortical (CCT) and medullary (MCT) collecting tubules of the rat nephron. At optimum conditions, NEM-sensitive ATPase activity was the same in both tubule segments: activity was 275.6 +/- 18.6 pmol/mm/h in the CCT and 280.3 +/- 35.2 pmol/mm/h in the MCT (n = 23, NS). ATP sensitivity was greater in CCT than in MCT, and in the former guanosine triphosphate was able to partially support enzyme activity. Maximal enzyme inhibition with NEM occurred at a lower concentration in CCT as compared to MCT. At pH 7.0 in MCT enzyme activity was approximately one half that seen at pH 7.4; in MCT and CCT, the pH optimum was 7.4. The temperature optimum in both segments was between 37 and 42 degrees C. Enzyme activity in CCT and MCT was linear to 30 min and proportional to tubule length. These results demonstrate that there are important differences in the NEM-sensitive ATPase isolated from two segments of rat collecting duct, and raise the possibility that enzyme heterogeneity may exist.

The effect of O-acetylsalicylic acid on lipid synthesis by guinea pig gastric mucosa in vitro.

The aim of this work was to investigate the involvement of lipids as possible components of the gastric mucosal barrier by studying the synthesis and secretion of lipids by the epithelial cell lining of gastric mucosa and the effect of salicylate on these processes. O-Acetylsalicylic acid reversibly reduced in vitro incorporation of [U-14C]acetate and of DL-[2-14C]mevalonic acid into lipids by isolated epithelial cells and by intact mucosa of guinea pig stomach, indicating reversible inhibition of lipid synthesis by the tissue in the presence of the drug. Inhibition of incorporation of both precursors into total lipids, into their fatty acid components, and into cholesterol is demonstrated.

Synthesis of IgA by human gastric mucosa in vitro.

Human gastric mucosa was removed at gastrectomy for duodenal ulcers. Intact mucosal layer and homogenized mucosal scrapings were incubated in vitro with L-[U-14C]leucine and with D-[U-14C]glucose and incorporation of radioactive label into IgA was investigated. 14C of both the precursors was incorporated into tissue IgA when intact gastric mucosa was incubated indicating synthesis of IgA by the tissue in vitro. IgA isolated from the media which bathed the luminal sides of the mucosae during incubation was also radioactive implying secretion of the newly-synthesized immunoglobulin into the media. When mucosal scrapings were incubated as homogenized suspensions, radioactivity of IgA was below the level of sensitivity of our methods. For analysis, IgA was separated from the bulk of radioactive mucosal proteins of the samples by isopycnic CsCl gradient centrifugation followed by gel filtration on Sephacryl S-200 or Sephadex G200 and then Sepharose 4B. The IgA-enriched Sepharose 4B fractions were subjected (1) to immunoprecipitation in agar gels by double diffusion against specific anti alpha-chain serum and (2) to SDS-polyacrylamide gel electrophoresis after reduction. Radioactivity of precipitated IgA, and of the protein band which corresponds to the alpha-chain of IgA on SDS-PAGE gels, was demonstrated autoradiographically. It is suggested that in vivo the mucosa is involved in local synthesis of the constituent IgA of gastric mucus.

Biosynthesis of proteins by human gastric mucosa in vitro.

Incorporation of L-[U-14C]leucine and of D[U-14C]glucose into proteins of fresh human gastric mucosa in vitro was studied after incubation of homogenized tissue and of intact mucosal pieces. CsCl centrifugation was used to separate high-density mucus glycoproteins from other mucosal proteins, and the macromolecular nature of radioactive mucosal glycoprotein fractions was confirmed by SDS/polyacrylamide-gel electrophoresis and autoradiography of the polyacrylamide gels. In all experiments a substantial proportion of total incorporated radioactivity was associated with gastric-mucosal glycoprotein fractions (CsCl fraction L3), indicating their biosynthesis. Radioactivity of these fractions was shown to co-chromatograph with carbohydrates when fractionated either directly or after reduction and alkylation (1) Sephadex G-200 chromatography in the excluded fractions and (2) by DEAE-cellulose ion-exchange chromatography. On incubation of intact mucosa, the major portion of radioactivity associated with the glycoprotein fractions of both leucine- and glucose-labelled specimens was secreted into the mucosal media during the course of the experiment. It is suggested that biosynthesis of mucus in vivo by gastric mucosa may be associated with rapid secretion of the synthesized macromolecules into the lumen of the stomach and that investigations of the metabolic processes within the mucosa should consider the products of secretion of the tissue. Incorporation of L-[U-14C]leucine implies biosynthesis of the polypeptide components of the macromolecules.

Ethanol, its effect on the synthesis of proteins by guinea-pig gastric mucosa.

Incorporation of L-[U-14C]leucine into proteins is taken to indicate the synthesis of proteins by guinea pig gastric mucosa. Ethanol reduced the synthesis of proteins in vitro by homogenized mucosa, by isolated gastric epithelial cell preparations and by intact tissue. Intact stomach wall incubated without ethanol in phosphate buffered saline showed progressively increasing incorporation of the precursor into tissue proteins and into proteins which were secreted into the mucosal incubation media. On isopycnic CsCl gradient fractionation radioactive tissue proteins were found at the top of the gradient (fraction L1, sp.gr.1.11-1.20) while radioactive secreted proteins sedimented to the bottom of the gradient as the carbohydrate rich high density gastric mucosal glycoprotein fraction L3 (sp.gr.1.29-1.33). Ethanol significantly but reversibly reduced the incorporation of radioactive leucine by intact mucosa into both the tissue proteins and the secreted proteins. Uptake of the precursor into the intracellular acid soluble pool was not impaired by ethanol and no significant differences were detected in the specific activities of free intracellular leucine between the ethanol treated samples and the corresponding controls. It is suggested that the ulcerogenic nature of ethanol may be associated with inhibition of the synthesis of proteins within mucosal epithelium leading to reduction in the output of mucosal secretory glycoproteins with subsequent impairment of the cytoprotective properties of the dynamic mucous barrier.

In vitro studies on the effect of salicylates on the synthesis of proteins by guinea pig gastric mucosal tissue.

In the work reported in this paper we have studied the effect of salicylates on protein synthesis by (A) intact guinea pig gastric mucosa, (B) isolated gastric epithelial cells of guinea pig stomach and (C) cell-free homogenates of the isolated cells. In experiments on intact gastric mucosa, (A), secretion of newly-synthesised proteins into the mucosal media was also investigated and the nature of the effected, secreted proteins examined by isopycnic CsCl gradient fractionation. Results indicate that synthesis of proteins, as assessed by incorporation of L-[U-14C]leucine into trichloroacetic acid-insoluble proteins, is significantly impaired in the presence of salicylates in all three systems investigated. In experiments using isolated epithelial cells and cell-free homogenates of these cells, the effect was found to be dose-dependent and not associated with a corresponding reduction of the uptake of the precursor from the medium into the acid-soluble intracellular pool. The inhibitory effect of salicylates on protein synthesis was found to be reversible in experiments using both the intact gastric mucosa, as also the isolated epithelial cell preparations. In all experiments it was found to last for the duration of exposure of the tissue to the action of the drug. In experiments using intact gastric mucosa secretion of the newly-synthesised radioactive proteins into the mucosal medium was also impaired by O-acetyl salicylic acid. Isopycnic CsCl gradient fractionation of the secreted proteins did not reveal any qualitative differences between the salicylate-treated samples and the control samples, indicating non-specific inhibition of protein synthesis by the drug.

Studies on human gastric mucosal immunoglobulin A.

Immunoglobulin A (IgA) was found in mucus scraped from the surface of the human antrum. Fresh human gastric mucosa removed at operation was washed free of loosely adhering material and the gelatinous mucus lining the tissue scraped. The scrapings were separated by gel filtration on Sephadex G-200 and on Sepharose 4B into two carbohydrate-containing fractions. One of these fractions was shown by immunodiffusion to contain IgA which differs from human colostral secretory IgA by being devoid of secretory component activity. Moreover, secretory component was not detected in our unfractionated gastric mucosal scrapings. It is concluded that, contrary to the general belief, the predominant immunoglobulin A of human gastric mucus is not associated with the secretory component. Our results do not exclude the possibility that, as in serum, small amounts of secretory IgA and of the secretory component may be present in gastric secretions, however if so, the levels of these compounds would fall below the level of sensitivity of our methods.

Cholesterol metabolism in myelin and other subcellular fractions of rat brain.

For many years the bulk of myelin in adult brain was believed to be metabolically stable, although some metabolic activity of a small myelin fraction, especially in the gray matter of the brain, was recognized. We have attempted to compare the composition of myelin fractions isolated from two different areas of the brain. No differences in chemical composition were observed. We have also investigated the metabolism of cholesterol in myelin and other subcellular fractions from the two areas. Both young (16-day-old) and adult rats were used. Results show an uptake of radioactive cholesterol by all subcellular fractions of the brain, including myelin, in both young and adult animals, with ultimate uniform distribution of the radioactive sterol and its persistence in all uniformly labeled subcellular fractions of the brain. On the basis of these results we suggest that there is a pool of cholesterol in the brain from which all metabolizing structures, including myelin, draw their cholesterol supplies. There is continuous exchange of cholesterol between the brain pool and the blood. The rate of this exchange may be related to the rate of blood flow through the tissue.

The identity of a myelin-like fraction isolated from developing brain.

1. A myelin-like membrane fraction was isolated from developing rat brain by a new method. 2. The chemical composition and morphology of the fraction are described. 3. The myelin-like fraction is similar to myelin in characteristic enzyme activity but differs in the absence of basic protein and cerebrosides. No similarity to other subcellular fractions was observed. 4. It is suggested that the myelin-like fraction is a stage in the formation of compact myelin from glial plasma membrane. 5. ;Early' myelin consists of the myelin-like and compact myelin fractions from developing brain.