Amplified B lymphocyte CD40 signaling drives regulatory B10 cell expansion in mice

Background

Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154^TG) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22^−/−) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154^TGCD22^−/− mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation.

Methodology/Principal Findings

CD154^TGCD22^−/− mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154^TGCD22^−/− mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154^TGCD22^−/− mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10⁶±6 in CD154^TGCD22^−/− mice; 1.7×10⁶±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10⁶±3 in CD154^TGCD22^−/− mice; 6.1×10⁶±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells.

Conclusions/Significance

These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.     

Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT _m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K_a = 16.2±0.6×10⁷ M⁻¹ where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K_a = 3.1±0.4×10⁷ M⁻¹) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.     

Genetic association study identifies HSPB7 as a risk gene for idiopathic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10⁻⁶, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10⁻⁵. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10⁻³, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10⁻³, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10⁻⁴, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10⁻¹³, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.     

The D299G/T399I Toll-like receptor 4 variant associates with body and liver fat: results from the TULIP and METSIM Studies

Background

Toll-like-receptor 4 (TLR) is discussed to provide a molecular link between obesity, inflammation and insulin resistance. Genetic studies with replications in non-diabetic individuals in regard to their fat distribution or insulin resistance according to their carrier status of a common toll-like receptor 4 (TLR4) variant (TLR4^D299G/T399I) are still lacking.

Methodology/Principal Findings

We performed a cross-sectional analysis in individuals phenotyped for prediabetic traits as body fat composition (including magnetic resonance imaging), blood glucose levels and insulin resistance (oral glucose tolerance testing, euglycemic hyperinsulinemic clamp), according to TLR4 genotype determined by candidate SNP analyses (rs4986790). We analyzed N = 1482 non-diabetic individuals from the TÜF/TULIP cohort (South Germany, aged 39±13 y, BMI 28.5±7.9, mean±SD) and N = 5327 non-diabetic participants of the METSIM study (Finland, males aged 58±6 y, BMI 26.8±3.8) for replication purposes. German TLR4^D299G/T399I carriers had a significantly increased body fat (XG in rs4986790: +6.98%, p = 0.03, dominant model, adjusted for age, gender) and decreased insulin sensitivity (XG: −15.3%, Matsuda model, p = 0.04; XG: −20.6%, p = 0.016, clamp; both dominant models adjusted for age, gender, body fat). In addition, both liver fat (AG: +49.7%; p = 0.002) and visceral adipose tissue (AG: +8.2%; p = 0.047, both adjusted for age, gender, body fat) were significantly increased in rs4986790 minor allele carriers, and the effect on liver fat remained significant also after additional adjustment for visceral fat (p = 0.014). The analysis in METSIM confirmed increased body fat content in association with the rare G allele in rs4986790 (AG: +1.26%, GG: +11.0%; p = 0.010, additive model, adjusted for age) and showed a non-significant trend towards decreased insulin sensitivity (AG: −0.99%, GG: −10.62%).

Conclusions/Significance

TLR4^D299G/T399I associates with increased total body fat, visceral fat, liver fat and decreased insulin sensitivity in non-diabetic Caucasians and may contribute to diabetes risk. This finding supports the role of TLR4 as a molecular link between obesity and insulin resistance.     

Toll-like receptor-2 mediates diet and/or pathogen associated atherosclerosis: proteomic findings

Background

Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE^+/− mice.

Methods and Results

To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE^+/−-TLR2^+/+, ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 µl live Porphyromonas gingivalis (P.g) (10⁷ CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE^+/−-TLR2^+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE^+/−-TLR2^+/+ mice were significantly higher than from ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE^+/−-TLR2^+/+ mice compared to ApoE^+/−-TLR2^+/− and TLR2^−/− mice, irrespective of diet or bacterial challenge. ApoE^+/−-TLR2^+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE^+/−-TLR2^−/− mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE^+/−-TLR2^+/+ mice fed the high fat diet and inoculated with P.g compared to any other group.

Conclusion

Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE^+/− mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.     

Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion

Purpose

To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.

Methods

Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-¹³C] glucose during an infusion of 6, 6-[²H₂] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.

Results

Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L⁻¹∶120 min, respectively) compared with CON (90.6±4.1 mmol·L⁻¹∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.

Conclusions

Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals.     

Apoptotic effects of antilymphocyte globulins on human pro-inflammatory CD4+CD28- T-cells

Background

Pro-inflammatory, cytotoxic CD4⁺CD28⁻ T-cells with known defects in apoptosis have been investigated as markers of premature immuno-senescence in various immune-mediated diseases. In this study we evaluated the influence of polyclonal antilymphocyte globulins (ATG-Fresenius, ATG-F) on CD4⁺CD28⁻ T-cells in vivo and in vitro.

Principal Findings

Surface and intracellular three colour fluorescence activated cell sorting analyses of peripheral blood mononuclear cells from 16 consecutive transplant recipients and short-term cell lines were performed. In vivo, peripheral levels of CD3⁺CD4⁺CD28⁻ T-cells decreased from 3.7±7.1% before to 0±0% six hours after ATG-F application (P = 0.043) in 5 ATG-F treated but not in 11 control patients (2.9±2.9% vs. 3.9±3.0%). In vitro, ATG-F induced apoptosis even in CD4⁺CD28⁻ T-cells, which was 4.3-times higher than in CD4⁺CD28⁺ T-cells. ATG-F evoked apoptosis was partially reversed by the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and prednisolon-21-hydrogensuccinate. ATG-F triggered CD25 expression and production of pro-inflammatory cytokines, and induced down-regulation of the type 1 chemokine receptors CXCR-3, CCR-5, CX3CR-1 and the central memory adhesion molecule CD62L predominately in CD4⁺CD28⁻ T-cells.

Conclusion

In summary, in vivo depletion of peripheral CD3⁺CD4⁺CD28⁻ T-cells by ATG-F in transplant recipients was paralleled in vitro by ATG-F induced apoptosis. CD25 expression and chemokine receptor down-regulation in CD4⁺CD28⁻ T-cells only partly explain the underlying mechanism.     

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺), led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.     

Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle

Background

Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.

Methodology/Principal Findings

We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.Oxidative phosphorylation (OXPHOS) capacity (pmol O₂•s⁻¹•mg⁻¹ wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness.

Conclusions/Significance

The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.     

Therapeutic trial of metformin and bortezomib in a mouse model of tuberous sclerosis complex (TSC)

Tuberous sclerosis complex (TSC) is a human genetic disorder in which loss of either TSC1 or TSC2 leads to development of hamartoma lesions, which can progress and be life-threatening or fatal. The TSC1/TSC2 protein complex regulates the state of activation of mTORC1. Tsc2^+/− mice develop renal cystadenoma lesions which grow progressively. Both bortezomib and metformin have been proposed as potential therapeutics in TSC. We examined the potential benefit of 1 month treatment with bortezomib, and 4 month treatment with metformin in Tsc2^+/− mice. Results were compared to vehicle treatment and treatment with the mTORC1 inhibitor rapamycin for 1 month. We used a quantitative tumor volume measurement on stained paraffin sections to assess the effect of these drugs. The median tumor volume per kidney was decreased by 99% in mice treated with rapamycin (p = 0.0004). In contrast, the median tumor volume per kidney was not significantly reduced for either the bortezomib cohort or the metformin cohort. Biochemical studies confirmed that bortezomib and metformin had their expected pharmacodynamic effects. We conclude that neither bortezomib nor metformin has significant benefit in this native Tsc2^+/− mouse model, which suggests limited benefit of these compounds in the treatment of TSC hamartomas and related lesions.     

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes

Background

Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes.

Methodology/Principal Findings

CD62P⁺ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14⁺CD16⁻, CD14^highCD16⁺and CD14^lowCD16⁺. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14^highCD16⁺ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P⁺-platelets (r² = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14⁺CD16⁻ cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14⁺CD16⁺ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r² = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809.

Conclusions/Significance

These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14^highCD16⁺ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.     

Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations

Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10⁻⁸) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (P _combined = 2.3×10⁻¹⁰, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10⁻¹⁰, OR = 1.52, and DPB1*0901: P = 2.0×10⁻⁷, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.     

In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

Genetic Modulation of Lipid Profiles following Lifestyle Modification or Metformin Treatment: The Diabetes Prevention Program

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10⁻¹⁷). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10⁻⁵–1×10⁻¹⁹). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10⁻⁵, P _interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P _interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P _interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P _interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.     

Functional energetics of CD4+-cellular immunity in monoclonal antibody-associated progressive multifocal leukoencephalopathy in autoimmune disorders

Background

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system- (CNS-) infection that typically occurs in a subset of immunocompromised individuals. An increasing incidence of PML has recently been reported in patients receiving monoclonal antibody (mAb) therapy for the treatment of autoimmune diseases, particularly those treated with natalizumab, efalizumab and rituximab. Intracellular CD4⁺-ATP-concentration (iATP) functionally reflects cellular immunocompetence and inversely correlates with risk of infections during immunosuppressive therapy. We investigated whether iATP may assist in individualized risk stratification for opportunistic infections during mAb-treatment.

Methodology/Principal Findings

iATP in PHA-stimulated, immunoselected CD4⁺-cells was analyzed using an FDA-approved assay. iATP of mAb-associated PML (natalizumab (n = 8), rituximab (n = 2), efalizumab (n = 1)), or other cases of opportunistic CNS-infections (HIV-associated PML (n = 2), spontaneous PML, PML in a psoriasis patient under fumaric acids, natalizumab-associated herpes simplex encephalitis (n = 1 each)) was reduced by 59% (194.5±29 ng/ml, mean±SEM) in comparison to healthy controls (HC, 479.9±19.8 ng/ml, p<0.0001). iATP in 14 of these 16 patients was at or below 3^rd percentile of healthy controls, similar to HIV-patients (n = 18). In contrast, CD4⁺-cell numbers were reduced in only 7 of 15 patients, for whom cell counts were available. iATP correlated with mitochondrial transmembrane potential (ΔΨ_m) (iATP/ΔΨ_m−correlation:tau = 0.49, p = 0.03). Whereas mean iATP of cross-sectionally analysed natalizumab-treated patients was unaltered (448.7±12 ng/ml, n = 150), iATP was moderately decreased (316.2±26.1 ng/ml, p = 0.04) in patients (n = 7) who had been treated already during the pivotal phase III trials and had received natalizumab for more than 6 years. 2/92 (2%) patients with less than 24 months natalizumab treatment revealed very low iATP at or below the 3^rd percentile of HC, whereas 10/58 (17%) of the patients treated for more than 24 months had such low iATP-concentrations.

Conclusion

Our results suggest that bioenergetic parameters such as iATP may assist in risk stratification under mAb-immunotherapy of autoimmune disorders.     

Adipocytokines and CD34 progenitor cells in Alzheimer's disease

Background

Alzheimer''s disease (AD) and atherosclerosis share common vascular risk factors such as arterial hypertension and hypercholesterolemia. Adipocytokines and CD34⁺ progenitor cells are associated with the progression and prognosis of atherosclerotic diseases. Their role in AD is not adequately elucidated.

Methods and Findings

In the present study, we measured in 41 patients with early AD and 37 age- and weight-matched healthy controls blood concentrations of adiponectin and leptin by enzyme linked immunoabsorbent assay and of CD34⁺ progenitor cells using flow cytometry. We found significantly lower plasma levels of leptin in AD patients compared with the controls, whereas plasma levels of adiponectin did not show any significant differences (AD vs. control (mean±SD): leptin:8.9±5.6 ng/mL vs.16.3±15.5 ng/mL;P = 0.038; adiponectin:18.5±18.1 µg/mL vs.16.7±8.9 µg/mL;P = 0.641). In contrast, circulating CD34⁺ cells were significantly upregulated in AD patients (mean absolute cell count±SD:253±51 vs. 203±37; P = 0.02) and showed an inverse correlation with plasma levels of leptin (r = −0.248; P = 0.037).In logistic regression analysis, decreased leptin concentration (P = 0.021) and increased number of CD34⁺ cells (P = 0.036) were both significantly associated with the presence of AD. According to multifactorial analysis of covariance, leptin serum levels were a significant independent predictor for the number of CD34⁺ cells (P = 0.002).

Conclusions

Our findings suggest that low plasma levels of leptin and increased numbers of CD34⁺ progenitor cells are both associated with AD. In addition, the results of our study provide first evidence that increased leptin plasma levels are associated with a reduced number of CD34⁺ progenitor cells in AD patients. These findings point towards a combined involvement of leptin and CD34⁺ progenitor cells in the pathogenesis of AD. Thus, plasma levels of leptin and circulating CD34⁺ progenitor cells could represent an important molecular link between atherosclerotic diseases and AD. Further studies should clarify the pathophysiological role of both adipocytokines and progenitor cells in AD and possible diagnostic and therapeutic applications.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Telomere shortening impairs regeneration of the olfactory epithelium in response to injury but not under homeostatic conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc^−/−) with short telomeres compared to wild type mice (mTerc^+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc^−/− mice compared to mTerc^+/+ mice. Seven days after chemical induced damage, G3 mTerc^−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc^+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people.     

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Background

Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).

Methodology/Principal Findings

We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.

Conclusion

Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.