CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages

CD8+ T lymphocytes have been implicated in the protective immune response against human and murine tuberculosis. However, the functional role that this cell subset plays during the resolution of infection remains controversial. In this study, we demonstrate the presence of Mycobacterium tuberculosis-specific CD8+ CTL in the lungs and lung-draining lymph nodes of mice infected with M. tuberculosis via the aerosol or i.v. route. These cells expressed perforin in vivo and specifically recognized and lysed M. tuberculosis-infected macrophages in a perforin-dependent manner after a short period of in vitro restimulation. The efficiency of lysis of infected macrophages was dependent upon the time allowed for interaction between macrophage and M. tuberculosis bacilli. Recognition of infected targets by CD8+ CTL was beta 2-microglobulin and MHC class I dependent and was not CD1d restricted. The presented data indicate that CD8+ T cells contribute to the protective immune response during M. tuberculosis infection by exerting cytotoxic function and lysing infected macrophages.     

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.     

Modelling Si-N-limited growth of diatoms

A mechanistic model for silicon (Si) physiology is developed,interfaced with a model of nitrogen (N) physiology, which iscapable of simulating the major documented facets of Si–Nphysiology in diatoms. The model contains a cell cycle componentthat is involved in regulating the timing of the synthesis ofvalves, girdles and setae. In addition to reproducing the timingof diatom cell division within a light–dark cycle, themodel simulates the following features seen in real diatoms.Synthesis of valves only occurs during G₂ interphase and M,while the girdles and (if appropriate) setae are synthesizedduring G₁. Si stress alone results in a loss of setae, followedby a thinning of the valves in successive generations untila minimum Si cell quota is attained. After this point, the durationof G₂ increases and growth is Si limited. Concurrently, thecarbon (C) cell quota increases, offering the capability tosimulate the documented increase in sinking rates with Si stress.N stress alone results in an increase in the duration of G₁and G₂ interphases, and high Si cell quotas. From this complexmodel, which must be run for arrays of subpopulations to simulatenon-synchronous growth, a simpler model is developed. This iscapable of reproducing similar growth dynamics, although withno reference to component parts of the frustule. When alliedto a photoacclimative submodel, a prediction of the model isthat diatoms starved of Si will release increased amounts ofdissolved organic C because cell growth is halted more rapidlythan the photosystems can be degraded.     

Participation of Vbeta13(+) and Vbeta1(+) T cells in transfer thyroiditis after activation of mouse thyroglobulin-primed T cells by superantigen staphylococcal enterotoxin A

Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of genes coding for cytokines, chemokines, and enzymes involved in amplification and perpetuation of inflammation. Hypoestoxide (a bicyclo [9,3,1] pentadecane) is a diterpene from Hypoestes rosea, a tropical shrub in the family Acanthacea, several members of which are used in folk medicine in Nigeria. Here, we demonstrate that hypoestoxide (HE) abrogates the production of pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated normal human peripheral blood mononuclear cells. Moreover, HE inhibits the production of nitric oxide (NO) by IL-1beta- or IL-17-stimulated normal human chondrocytes. In vivo, oral administration of HE to mice significantly ameliorated hind paw edema induced by antibodies to type II collagen plus LPS. Furthermore, topical administration of HE to mice also significantly inhibited phorbol ester-induced ear inflammation. The anti-inflammatory activity of HE may be due in part to its ability to inhibit NF-kappaB activation through direct inhibition of IkappaB kinase (IKK) activity. Thus, HE could be useful in treating various inflammatory diseases and may represent a prototype of a novel class of IKK inhibitors.     

Domain effects on the DNA-interactive properties of bacteriophage T4 gene 32 protein

Bacteriophage T4 gene 32 protein, a model for single-strand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can "mingle" with intact protein or core domain while bound to single-stranded DNA.     

N-1 substituted pyrimidin-4-ones: novel, orally active inhibitors of lipoprotein-associated phospholipase A2

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.     

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.     

Field records of painted apple moth (Teia anartoides Walker: Lepidoptera: Lymantriidae) on plants and inanimate objects in Auckland, New Zealand

Abstract  As part of an eradication program, field searches were conducted to verify the geographical extent of the painted apple moth (PAM; Teia anartoides Walker: Lepidoptera: Lymantriidae) incursion and to determine which plants were being attacked. Most PAM were found on the wattle species, in particular Paraserianthes lophanta and Acacia mearnsii . Other species with large numbers of PAM included the native Corynocarpus laevigatus (karaka) and Avicennia marina (grey mangrove). The large number of PAM on these New Zealand species would not have been predicted from the known unrelated Australian hosts. The relatively high number of PAM found on inanimate objects demonstrated the mobility of PAM larvae and that plants upon which few larvae were found may not be suitable hosts for PAM.     

Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion

Background

Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool.

The effect of changes to GOLD severity stage on long term morbidity and mortality in COPD

Background

The Global Initiative for Chronic Obstructive Lung Disease (GOLD) severity stage classifies Chronic Obstructive Pulmonary Disease (COPD) into groups based on symptoms, exacerbations and forced expiratory volume in one second (FEV₁). This allows patients to change to less severe COPD stages, a novel aspect of assessment not previously evaluated. We aimed to investigate the association between temporal changes in GOLD severity stage and outcomes in COPD patients.

Methods

This was a record-linkage study using patients registered with a Scottish regional COPD network 2000–2015. Annual spirometry & symptoms were recorded and linked to healthcare records to identify exacerbations, hospitalisations and mortality. Spirometry, modified Medical Research Council (mMRC) dyspnoea scale and acute exacerbations over the previous year were used to assign GOLD severity at each visit. A time-dependent Cox model was used to model time to death. Secondary outcomes were respiratory specific mortality and hospitalisations. Effect sizes are expressed as Hazard Ratios HR (95%CI).

Results

Four thousand, eight hundred and eighty-five patients (mean age 67.3?years; 51.3% female) with 21,348 visits were included. During a median 6.6?years follow-up there were 1530 deaths. For the secondary outcomes there were 712 respiratory deaths and 1629 first hospitalisations. Across 16,463 visit-pairs, improvement in COPD severity was seen in 2308 (14%), no change in 11,010 (66.9%) and worsening in 3145 (19.1). Compared to patients staying in GOLD stage A, those worsening had a stepwise increased mortality and hospitalisations.

Conclusions

Improving COPD severity classification was associated with reduced mortality and worsening COPD severity was associated with increased mortality and hospitalisations. Change in GOLD group has potential as monitoring tool and outcome measure in clinical trials.