Anti-tumor necrosis factor therapy improves insulin resistance, beta cell function and insulin signaling in active rheumatoid arthritis patients with high insulin resistance

Introduction

Prevalence of insulin resistance and the metabolic syndrome has been reported to be high in rheumatoid arthritis (RA) patients. Tumor necrosis factor (TNF), a pro-inflammatory cytokine with a major pathogenetic role in RA, may promote insulin resistance by inducing Ser³¹² phosphorylation (p-Ser³¹²) of insulin receptor substrate (IRS)-1 and downregulating phosphorylated (p-)AKT. We examined whether anti-TNF therapy improves insulin resistance in RA patients and assessed changes in the insulin signaling cascade.

Methods

Prospective study of RA patients receiving anti-TNF agents (infliximab, n = 49, adalimumab, n = 11, or etanercept, n = 1) due to high disease activity score in 28 joints (DAS28 > 5.1). A complete biochemical profile was obtained at weeks 0 and 12 of treatment. Insulin resistance, insulin sensitivity and pancreatic beta cell function were measured by the Homeostasis Model Assessment (HOMA-IR), the Quantitative Insulin Sensitivity Check Index (QUICKI) and the HOMA-B respectively. Protein extracts from peripheral blood mononuclear cells were assayed by western blot for p-Ser³¹² IRS-1 and p-AKT. RA patients treated with abatacept (CTLA4.Ig) were used as a control group for insulin signaling studies.

Results

At study entry, RA patients with high insulin resistance (HOMA-IR above median) had significantly higher mean DAS28 (P = 0.011), serum triglycerides (P = 0.015), and systolic blood pressure levels (P = 0.024) than patients with low insulin resistance. After 12 weeks of anti-TNF therapy, patients with high insulin resistance demonstrated significant reduction in HOMA-IR (P < 0.001), HOMA-B (P = 0.001), serum triglycerides (P = 0.039), and increase in QUICKI (P < 0.001) and serum HDL-C (P = 0.022). Western blot analysis in seven active RA patients with high insulin resistance showed reduction in p-Ser³¹² IRS-1 (P = 0.043) and increase in p-AKT (P = 0.001) over the study period. In contrast, the effect of CTLA4.Ig on p-Ser³¹² IRS-1 and p-AKT levels was variable.

Conclusions

Anti-TNF therapy improved insulin sensitivity and reversed defects in the insulin signaling cascade in RA patients with active disease and high insulin resistance. The impact of these biochemical changes in modifying cardiovascular disease burden in active RA patients remains to be seen.     

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L.?monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (?thiT). Cells of both wild-type and ?thiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.     

The role of phase separation and feed cycle length in leach beds coupled to methanogenic reactors for digestion of a solid substrate (Part 1): Optimisation of reactors' performance

A two-phase system composed by a leach bed and a methanogenic reactor was modified for the first time to improve volumetric substrate degradation and methane yields from a complex substrate (maize; Zeamays). The system, which was operated for consecutive feed cycles of different durations for 120 days, was highly flexible and its performance improved by altering operational conditions. Daily substrate degradation was higher the shorter the feed cycle, reaching 8.5 g TS_destroyed d⁻¹ (7-day feed cycle) but the overall substrate degradation was higher by up to 55% when longer feed cycles (14 and 28 days) were applied. The same occurred with volumetric methane yields, reaching 0.839 m³ (m³)⁻¹ d⁻¹. The system performed better than others on specific methane yields, reaching 0.434 m³ kg⁻¹ TS_added, in the 14-day and 28-day systems. The UASB and AF designs performed similarly as second stage reactors on methane yields, SCOD and VFA removal efficiencies.     

Genetic polymorphisms within tumor necrosis factor gene promoter region: a role for susceptibility to ventilator-associated pneumonia

Debatable findings exist among various studies regarding the impact of single nucleotide polymorphisms (SNPs) within the promoter region of the tumor necrosis factor (TNF) gene for susceptibility to infections. Their impact was investigated in a cohort of mechanically ventilated patients who developed ventilator-associated pneumonia (VAP). Two-hundred and thirteen mechanically ventilated patients who developed VAP were enrolled. Genomic DNA was extracted and SNPs at the -376, -308 and -238 position of the promoter region of the TNF gene were assessed by restriction fragment length polymorphisms. Monocytes were isolated from 47 patients when they developed sepsis and stimulated by bacterial endotoxin for the production of TNFα and of interleukin-6 (IL-6). Patients were divided into two groups; 166 patients bearing only wild-type alleles of all three studied polymorphisms; and 47 patients carrying at least one A allele of the three studied SNPs. Time between start of mechanical ventilation and advent of VAP was significantly shorter in the second group than in the first group (log-rank: 4.416, p: 0.041). When VAP supervened, disease severity did not differ between groups. Stimulation of TNFα and of IL-6 was much greater by monocytes for patients carrying A alleles. Carriage of at least one A allele of the three studied SNPs at the promoter region of the TNF-gene is associated with shorter time to development of VAP but it is not associated with disease severity. Findings may be related with a role of the studied SNPs in the production of pro-inflammatory cytokines.     

Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing

Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb) and 7 (1.1 Mb) from an individual from the International HapMap Project (NA12872). We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage≥4-fold, and 97.9% concordant in regions with coverage≥15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.     

Genome scan meta-analysis of schizophrenia and bipolar disorder,part II: Schizophrenia

Schizophrenia is a common disorder with high heritability and a 10-fold increase in risk to siblings of probands. Replication has been inconsistent for reports of significant genetic linkage. To assess evidence for linkage across studies, rank-based genome scan meta-analysis (GSMA) was applied to data from 20 schizophrenia genome scans. Each marker for each scan was assigned to 1 of 120 30-cM bins, with the bins ranked by linkage scores (1 = most significant) and the ranks averaged across studies (R(avg)) and then weighted for sample size (N(sqrt)[affected casess]). A permutation test was used to compute the probability of observing, by chance, each bin's average rank (P(AvgRnk)) or of observing it for a bin with the same place (first, second, etc.) in the order of average ranks in each permutation (P(ord)). The GSMA produced significant genomewide evidence for linkage on chromosome 2q (PAvgRnk<.000417). Two aggregate criteria for linkage were also met (clusters of nominally significant P values that did not occur in 1,000 replicates of the entire data set with no linkage present): 12 consecutive bins with both P(AvgRnk) and P(ord)<.05, including regions of chromosomes 5q, 3p, 11q, 6p, 1q, 22q, 8p, 20q, and 14p, and 19 consecutive bins with P(ord)<.05, additionally including regions of chromosomes 16q, 18q, 10p, 15q, 6q, and 17q. There is greater consistency of linkage results across studies than has been previously recognized. The results suggest that some or all of these regions contain loci that increase susceptibility to schizophrenia in diverse populations.     

Effect of CDP-choline on hippocampal acetylcholinesterase and Na+,K(+)-ATPase in adult and aged rats

The aim of this study was to investigate the effect of different cytidine-5'-diphosphocholine (CDP-choline) concentrations (0.1-1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg(2+)-ATPase activities in homogenates of adult and aged rat hippocampi. Tissues were homogenised, centrifuged at 1000 x g for 10 min and in the supernatant, AChE activity and Na+,K(+)-ATPase and Mg(2+)-ATPase activities were determined according to Ellman's method and Bowler's and Tirri's method, respectively. After an 1-3 h preincubation of the homogenised tissue with CDP-choline, a maximal AChE stimulation of about 25% for both adult and aged rats (p < 0.001) and a Na+,K(+)-ATPase activation of about 50% for adult rats (p < 0.001) and about 60% for aged rats (p < 0.001) were observed, while hippocampal Mg(2+)-ATPase activity was not influenced in either adult or aged animals. It is suggested that: CDP-choline can restore hippocampal AChE and Na+,K(+)-ATPase activities in the aged rat and thus it may play a role in improving memory performance which is impaired by aging and some neuronal disturbances.     

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance,stress resistance,motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.     

ULTRASTRUCTURE OF CARPOSPOROPHYTE DEVELOPMENT IN THE RED ALGA GLOIOSIPHONIA VERTICILLARIS (CRYPTONEMIALES,GLOIOSIPHONIACEAE)

The ultrastructure of carposporophyte development is described for the red alga Gloiosiphonia verticillaris Farl. The auxiliary cell produces gonimoblast initials, which divide to produce two types of gonimoblast cells—the nondividing vacuolate cells and terminal generative gonimoblast cells. The generative gonimoblast cells form clusters of carpospore initials, which eventually differentiate into carpospores. After gonimoblast filaments are formed, the auxiliary cell undergoes autolysis, causing degeneration of septal plugs between the auxiliary cell and adjacent cells, thus forming a fusion cell. Since this cell lacks starch and appears degenerate throughout carposporophyte development, a nutritive function cannot be ascribed to the fusion cell. Carpospore differentiation is simple and proceeds through three developmental stages. Young carpospores structurally resemble gonimoblast cells, because they contain undeveloped plastids, large quantities of floridean starch, and are surrounded by extensive mucilage instead of a distinct wall. In addition, dictyosomes form and begin to produce vesicles with fibrous contents representing carpospore wall material. During the intermediate stage, dictyosomes continue to produce vesicles that contribute additional carpospore wall material, thereby compressing the mucilage and creating a darker-staining layer outside the carpospore wall. Plastids form internal thylakoids by invaginations of the inner membrane of the peripheral thylakoid. The endoplasmic reticulum forms large granular vacuoles that appear to be degraded during subsequent stages of development. Mature carpospores form cored vesicles. They also contain mature chloroplasts, large amounts of floridean starch, and occasionally granular vacuoles. During this stage, interconnecting carpospore-carpospore and carpospore-gonimoblast cell septal plugs begin to undergo degeneration. This process may be mediated by tubular structures.