IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses,Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG₁ anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG₁, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG₄ peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG₄, the ACPA-IgG₄ Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG₁ compared to FT-IgG₁ were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG₁ relative to FT-IgG₁ as well as lower content of IgG₂ (p = 0.0000001) and elevated content of IgG₄ (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG₁ and ACPA-IgG₄ are distinct. Given that IgG₁ and IgG₄ have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.     

Molecular Characterization of Klebsiella pneumoniae Carbapenemase (KPC)-Producing Enterobacteriaceae in Ontario,Canada, 2008-2011

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of bla _KPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as bla _KPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored bla _KPC-2 (n = 23) or bla _KPC-3 (n = 7). bla _TEM-1 (n = 27) was commonly detected and occasionally bla _OXA-1 (n = 3) and bla _CTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried bla _SHV-11. bla _KPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.     

Association of Germline Variation in CCNE1 and CDK2 with Breast Cancer Risk,Progression and Survival among Chinese Han Women

Background

Somatic alterations of cyclin-dependent kinase 2 (CDK2)-cyclin E complex have been shown to contribute to breast cancer (BC) development and progression. This study aimed to explore the effects of single nucleotide polymorphisms (SNPs) in CDK2 and CCNE1 (a gene encoding G1/S specific cyclin E1 protein, formerly called cyclin E) on BC risk, progression and survival in a Chinese Han population.

Methodology/Principal Findings

We herein genotyped 6 haplotype-tagging SNPs (htSNPs) of CCNE1 and 2 htSNPs of CDK2 in 1207 BC cases and 1207 age-matched controls among Chinese Han women, and then reconstructed haplotype blocks according to our genotyping data and linkage disequilibrium status of these htSNPs. For CCNE1, the minor allele homozygotes of three htSNPs were associated with BC risk (rs3218035: adjusted odds ratio [aOR] = 3.35, 95% confidence interval [CI] = 1.69–6.67; rs3218038: aOR = 1.81, 95% CI = 1.22–2.70; rs3218042: aOR = 2.64, 95% CI = 1.31–5.34), and these three loci showed a dose-dependent manner in increasing BC risk (P _trend = 0.0001). Moreover, the 5-SNP haplotype CCGTC, which carried none of minor alleles of the 3 at-risk SNPs, was associated with a favorable event-free survival (hazard ratio [HR] = 0.53, 95% CI = 0.32–0.90). Stratified analysis suggested that the minor-allele homozygote carriers of rs3218038 had a worse event-free survival among patients with aggressive tumours (in tumour size>2 cm group: HR = 2.06, 95% CI = 1.06–3.99; in positive lymph node metastasis group: HR = 2.41, 95% CI = 1.15–5.03; in stage II–IV group: HR = 2.03, 95% CI = 1.09–3.79). For CDK2, no significant association was found.

Conclusions/Significance

This study indicates that genetic variants in CCNE1 may contribute to BC risk and survival in Chinese Han population. They may become molecular markers for individual evaluation of BC susceptibility and prognosis. Nevertheless, further validation studies are needed.     

Genetic Susceptibility to Refractive Error: Association of Vasoactive Intestinal Peptide Receptor 2 (VIPR2) with High Myopia in Chinese

Myopia is the most common ocular disease worldwide. We investigated the association of high myopia with the common single nucleotide polymorphisms (SNPs) of five candidate genes – early growth response 1 (EGR1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), jun oncogene (JUN), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide receptor 2 (VIPR2). We recruited 1200 unrelated Chinese subjects with 600 cases (spherical equivalent ≤−8.00 diopters) and 600 controls (spherical equivalent within ±1.00 diopter). A discovery sample set was formed from 300 cases and 300 controls, and a replication sample set from the remaining samples. Tag SNPs were genotyped for the discovery sample set, and the most significant haplotypes and their constituent SNPs were followed up with the replication sample set. The allele and haplotype frequencies in cases and controls were compared by logistic regression adjusted for sex and age to give P _a values, and multiple comparisons were corrected by permutation test to give P _aemp values. Odd ratios (OR) were calculated accordingly. In the discovery phase, EGR1, JUN and VIP did not show any significant association while FOS and VIPR2 demonstrated significant haplotype association with high myopia. In the replication phase, the haplotype association for VIPR2 was successfully replicated, but not FOS. In analysis combining both sample sets, the most significant association signals of VIPR2 were the single marker rs2071625 (P _a = 0.0008, P _aemp = 0.0046 and OR = 0.75) and the 4-SNP haplotype window rs2071623-rs2071625-rs2730220-rs885863 (omnibus test, P _a = 9.10e-10 and P _aemp = 0.0001) with one protective haplotype (GGGG: P _aemp = 0.0002 and OR = 0.52) and one high-risk haplotype (GAGA: P _aemp = 0.0027 and OR = 4.68). This 4-SNP haplotype window was the most significant in all sample sets examined. This is the first study to suggest a role of VIPR2 in the genetic susceptibility to high myopia. EGR1, JUN, FOS and VIP are unlikely to be important in predisposing humans to high myopia.     

Diagnosis of Bladder Cancer Recurrence Based on Urinary Levels of EOMES,HOXA9, POU4F2, TWIST1, VIM,and ZNF154 Hypermethylation

Background

Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine.

Methodology/Principal Findings

The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%–89% and specificity in the range 94%–100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88–94% and specificities of 43–67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P≤0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR = 7.8, P = 0.006), POU4F2 (HR = 8.5, P = 0.001), TWIST1 (HR = 12.0, P = 0.015), VIM (HR = 8.0, P = 0.001), and ZNF154 (HR = 13.9, P<0.001). Interestingly, for one group of patients (n = 15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect.

Conclusion/Significance

Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.     

Mitochondrial Common Deletion,a Potential Biomarker for Cancer Occurrence,Is Selected against in Cancer Background: A Meta-Analysis of 38 Studies

Mitochondrial dysfunction has been long proposed to play a major role in tumorigenesis. Mitochondrial DNA (mtDNA) mutations, especially the mtDNA 4,977 bp deletion has been found in patients of various types of cancer. In order to comprehend the mtDNA 4,977 bp deletion status in various cancer types, we performed a meta-analysis composed of 33 publications, in which a total of 1613 cancer cases, 1516 adjacent normals and 638 healthy controls were included. When all studies were pooled, we found that cancerous tissue carried a lower mtDNA 4,977 bp deletion frequency than adjacent non-cancerous tissue (OR = 0.43, 95% CI = 0.20–0.92, P = 0.03 for heterogeneity test, I² = 91.5%) among various types of cancer. In the stratified analysis by cancer type the deletion frequency was even lower in tumor tissue than in adjacent normal tissue of breast cancer (OR = 0.19, 95% CI = 0.06–0.61, P = 0.005 for heterogeneity test, I² = 82.7%). Interestingly, this observation became more significant in the stratified studies with larger sample sizes (OR = 0.70, 95% CI = 0.58–0.86, P = 0.0005 for heterogeneity test, I² = 95.1%). Furthermore, when compared with the normal tissue from the matched healthy controls, increased deletion frequencies were observed in both adjacent non-cancerous tissue (OR = 3.02, 95% CI = 2.13–4.28, P<0.00001 for heterogeneity test, I² = 53.7%), and cancerous tissue (OR = 1.36, 95% CI = 1.04–1.77, P = 0.02 for heterogeneity test, I² = 83.5%). This meta-analysis suggests that the mtDNA 4,977 bp deletion is often found in cancerous tissue and thus has the potential to be a biomarker for cancer occurrence in the tissue, but at the same time being selected against in various types of carcinoma tissues. Larger and better-designed studies are still warranted to confirm these findings.     

Immunization against a Saccharide Epitope Accelerates Clearance of Experimental Gonococcal Infection

The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a T_H1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with currently available antibiotics.     

Genetic Polymorphisms of GSTM1, GSTT1, and GSTP1 with Prostate Cancer Risk: A Meta-Analysis of 57 Studies

Background and Objectives

The GSTM1, GSTT1 and GSTP1 polymorphisms might be involved in inactivation of procarcinogens that contribute to the genesis and progression of cancers. However, studies investigating the association between GSTM1, GSTT1 or GSTP1 polymorphisms and prostate cancer (PCa) risk report conflicting results, therefore, we conducted a meta-analysis to re-examine the controversy.

Methods

Published literature from PubMed, Embase, Google Scholar and China National Knowledge Infrastructure (CNKI) were searched (updated to June 2, 2012). According to our inclusion criteria, studies that observed the association between GSTM1, GSTT1 or GSTP1 polymorphisms and PCa risk were included. The principal outcome measure was the odds ratio (OR) with 95% confidence interval (CI) for the risk of PCa associated with GSTM1, GSTT1 and GSTP1 polymorphisms.

Results

Fifty-seven studies involving 11313 cases and 12934 controls were recruited. The overall OR, which was 1.2854 (95% CI = 1.1405–1.4487), revealed a significant risk of PCa and GSTM1 null genotype, and the similar results were observed when stratified by ethnicity and control source. Further, the more important is that the present study first reported the high risks of PCa for people who with dual null genotype of GSTM1 and GSTT1 (OR = 1.4353, 95% CI = 1.0345–1.9913), or who with GSTT1 null genotype and GSTP1 A131G polymorphism (OR = 1.7335, 95% CI = 1.1067–2.7152). But no association was determined between GSTT1 null genotype (OR = 1.102, 95% CI = 0.9596–1.2655) or GSTP1 A131G polymorphism (OR = 1.0845, 95% CI = 0.96–1.2251) and the PCa risk.

Conclusions

Our meta-analysis suggested that the people with GSTM1 null genotype, with dual null genotype of GSTM1 and GSTT1, or with GSTT1 null genotype and GSTP1 A131G polymorphism are associated with high risks of PCa, but no association was found between GSTT1 null genotype or GSTP1 A131G polymorphism and the risk of PCa. Further rigorous analytical studies are highly expected to confirm our conclusions and assess gene-environment interactions with PCa risk.     

Association between Body Water Status and Acute Mountain Sickness

Purpose

The present study determined the association between body fluid variation and the development of acute mountain sickness (AMS) in adults.

Methods

Forty-three healthy participants (26 males and 17 females, age: 26±6 yr, height: 174±9 cm, weight: 68±12 kg) were passively exposed at a FiO₂ of 12.6% (simulated altitude hypoxia of 4500 m, PiO₂ = 83.9 mmHg) for 12-h. AMS severity was assessed using the Lake Louise Score (LLS). Food and drink intakes were consumed ad libitum and measured; all urine was collected. Before and after the 12-h exposure, body weight and plasma osmolality were measured and whole-body bioimpedance analysis was performed.

Results

The overall AMS incidence was 43% (38% males, 50% females). Participants who developed AMS showed lower fluid losses (3.0±0.9 vs. 4.5±2.0 ml/kg/h, p = 0.002), a higher fluid retention (1.9±1.5 vs. 0.6±0.8 ml/kg/h, p = 0.022), greater plasma osmolality decreases (−7±7 vs. −2±5 mOsm/kg, p = 0.028) and a larger plasma volume expansion (11±10 vs. 1±15%, p = 0.041) compared to participants not developing AMS. Net water balance (fluid intake – fluid loss) and the amount of fluid loss were strong predictors whether getting sick or not (Nagelkerkes r² = 0.532). The LLS score was related to net water balance (r = 0.358, p = 0.018), changes in plasma osmolality (r = −0.325, p = 0.033) and sodium concentration (r = −0.305, p = 0.047). Changes in the impedance vector length were related to weight changes (r = −0.550, p<0.001), fluid intake (r = −0.533, p<0.001) and net water balance (r = −0.590, p<0.001).

Conclusions

Participants developing AMS within 12 hours showed a positive net water balance due to low fluid loss. Thus measures to avoid excess fluid retention are likely to reduce AMS symptoms.     

Developmental Dysplasia of the Hip,Age, BMI,Place of Residence and Tobacco Abuse Increase the Odds of Aseptic Loosening in Chinese Patients

Purpose

The purpose of this hospital-based case-control study was to evaluate the patient-related risk factors for aseptic loosening after total hip arthroplasty (THA) and total knee arthroplasty (TKA) in Chinese patients.

Methods

From January 2000 to December 2012, 67 patients undergoing THA and TKA who developed aseptic loosening were detected as case subjects and 336 patients without aseptic loosening, matched by the year of index surgery and type of surgery, were selected as controls. Conditional logistic regression was used to compute odds ratios (ORs) and 95% confidence intervals (CIs).

Results

The demographic factors and comorbid conditions associated with a risk-adjusted increase in aseptic loosening (in decreasing order of significance) were a rural place of residence (OR = 2.28; 95% CI: 1.21–4.30; p = 0.011), body mass index (BMI) ≥28 kg/m² (vs. 18.5–28 kg/m²) (OR = 2.29; 95% CI: 1.19–4.41; p = 0.013), developmental dysplasia of the hip (DDH) (OR = 2.91; 95% CI: 1.11–7.66; p = 0.030), tobacco abuse (OR = 2.88; 95% CI: 1.05–7.89; p = 0.039), and age <45 years (vs. 45–65 years) (OR = 2.63; 95% CI: 1.01–6.80; p = 0.047).

Conclusions

Patients aged <45 years and those with a BMI of ≥28 kg/m², a preoperative diagnosis of DDH, history of tobacco abuse, or living in rural areas are at increased risk for aseptic loosening after THA and TKA in Chinese population. Additional systematic large-scale studies are needed to verify these results.     

Does the KIR2DS5 Gene Protect from Some Human Diseases?

Background

KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation.

Methodology/Principal Findings

In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p = 0.003, odds ratio [OR] = 0.47, confidence interval [CI] = 0.28–0.79), endometriosis (p = 0.03, OR = 0.25, CI = 0.07–0.82) and acute rejection of kidney graft (p = 0.0056, OR = 0.44, CI = 0.24–0.80), but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05). In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p = 0.0003, OR = 0.35, CI = 0.19–0.65), whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p = 0.0017, OR = 1.92, CI = 1.28–2.89), whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p = 0.005, OR = 1.47, CI = 1.13–1.92). The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p = 0.017, OR = 0.47, CI = 0.25–0.89), whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p = 0.0015, OR = 2.13, CI = 1.34–3.37).

Conclusions/Significance

Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.     

Investigation of Type 2 Diabetes Risk Alleles Support CDKN2A/B,CDKAL1, and TCF7L2 As Susceptibility Genes in a Han Chinese Cohort

Background

Recent genome-wide association studies (GWASs) have reported several genetic variants to be reproducibly associated with type 2 diabetes. Additional variants have also been detected from a metaanalysis of three GWASs, performed in populations of European ancestry. In the present study, we evaluated the influence of 17 genetic variants from 15 candidate loci, identified in type 2 diabetes GWASs and the metaanalysis, in a Han Chinese cohort.

Methodology/Principal Findings

Selected type 2 diabetes–associated genetic variants were genotyped in 1,165 type 2 diabetic patients and 1,136 normoglycemic control individuals of Southern Han Chinese ancestry. The OR for risk of developing type 2 diabetes was calculated using a logistic regression model adjusted for age, sex, and BMI. Genotype-phenotype associations were tested using a multivariate linear regression model. Genetic variants in CDKN2A/B, CDKAL1, TCF7L2, TCF2, MC4R, and PPARG showed a nominal association with type 2 diabetes (P≤0.05), of whom the three first would stand correction for multiple testing: CDKN2A/B rs10811661, OR: 1.26 (1.12–1.43) P = 1.8*10⁻⁴; CDKAL1 rs10946398, OR: 1.23 (1.09–1.39); P = 7.1*10⁻⁴, and TCF7L2 rs7903146, OR: 1.61 (1.19–2.18) P = 2.3 * 10⁻³. Only nominal phenotype associations were observed, notably for rs8050136 in FTO and fasting plasma glucose (P = 0.002), postprandial plasma glucose (P = 0.002), and fasting C-peptide levels (P = 0.006) in the diabetic patients, and with BMI in controls (P = 0.033).

Conclusions/Significance

We have identified significant association between variants in CDKN2A/B, CDKAL1 and TCF7L2, and type 2 diabetes in a Han Chinese cohort, indicating these genes as strong candidates conferring susceptibility to type 2 diabetes across different ethnicities.     

Evidence from a Randomized Trial That Simvastatin,but Not Ezetimibe,Upregulates Circulating PCSK9 Levels

Background

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted inhibitor of the low-density lipoprotein (LDL) receptor and an important regulator of LDL metabolism. Elevated PCSK9 levels have been associated with cardiovascular risk. The purpose of this study was to investigate how ezetimibe and simvastatin, alone and in combination, affect PCSK9 circulating concentrations.

Methods

A single center, randomized, open-label parallel 3-group study in healthy men (mean age 32±9 years, body mass index 25.7±3.2 kg/m²) was performed. Each group of 24 subjects was treated for 14 days with either simvastatin 40 mg/d, ezetimibe 10 mg/d, or with both drugs. Multivariate analysis was used to investigate parameters influencing the change in PCSK9 concentrations under treatment.

Results

The baseline plasma PCSK9 concentrations in the total cohort were 52±20 ng/mL with no statistically significant differences between the groups. They were increased by 68±85% by simvastatin (P = 0.0014), by 10±38% by ezetimibe (P = 0.51) and by 67±91% by simvastatin plus ezetimibe (P = 0.0013). The increase in PCSK9 was inversely correlated with baseline PCSK9 concentrations (Spearman’s R = –0.47, P<0.0001) and with the percent change in LDL cholesterol concentrations (Spearman’s R = –0.30, P<0.01). In multivariate analyses, only baseline PCSK9 concentrations (β = –1.68, t = –4.04, P<0.0001), percent change in LDL cholesterol from baseline (β = 1.94, t = 2.52, P = 0.014), and treatment with simvastatin (P = 0.016), but not ezetimibe (P = 0.42), significantly influenced changes in PCSK9 levels. Parameters without effect on PCSK9 concentration changes were age, body mass index, body composition, thyroid function, kidney function, glucose metabolism parameters, adipokines, markers of cholesterol synthesis and absorption, and molecular markers of cholesterol metabolism.

Conclusions

Ezetimibe does not increase circulating PCSK9 concentrations while simvastatin does. When added to simvastatin, ezetimibe does not cause an incremental increase in PCSK9 concentrations. Changes in PCSK9 concentrations are tightly regulated and mainly influenced by baseline PCSK9 levels and changes in LDL cholesterol.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00317993","term_id":"NCT00317993"}}NCT00317993     

Role of Peptidergic Nerve Terminals in the Skin: Reversal of Thermal Sensation by Calcitonin Gene-Related Peptide in TRPV1-Depleted Neuropathy

To investigate the contribution of peptidergic intraepidermal nerve fibers (IENFs) to nociceptive responses after depletion of the thermal-sensitive receptor, transient receptor potential vanilloid subtype 1 (TRPV1), we took advantage of a resiniferatoxin (RTX)-induced neuropathy which specifically affected small-diameter dorsal root ganglion (DRG) neurons and their corresponding nerve terminals in the skin. Thermal hypoalgesia (p<0.001) developed from RTX-treatment day 7 (RTXd7) and became normalized from RTXd56 to RTXd84. Substance P (SP)(+) and TRPV1(+) neurons were completely depleted (p = 0.0001 and p<0.0001, respectively), but RTX had a relatively minor effect on calcitonin gene-related peptide (CGRP)(+) neurons (p = 0.029). Accordingly, SP(+) (p<0.0001) and TRPV1(+) (p = 0.0008) IENFs were permanently depleted, but CGRP(+) IENFs (p = 0.012) were only transiently reduced and had recovered by RTXd84 (p = 0.83). The different effects of RTX on peptidergic neurons were attributed to the higher co-localization ratio of TRPV1/SP than of TRPV1/CGRP (p = 0.029). Thermal hypoalgesia (p = 0.0018) reappeared with an intraplantar injection of botulinum toxin type A (botox), and the temporal course of withdrawal latencies in the hot-plate test paralleled the innervation of CGRP(+) IENFs (p = 0.0003) and CGRP contents in skin (p = 0.01). In summary, this study demonstrated the preferential effects of RTX on depletion of SP(+) IENFs which caused thermal hypoalgesia. In contrast, the skin was reinnervated by CGRP(+) IENFs, which resulted in a normalization of nociceptive functions.     

Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells,Mice, and Humans

Background

FK506 binding protein 51 (FKBP51) is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.

Methods and Findings

Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO) mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR) treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways.Importantly, the clinical antidepressant response of patients with depression (n = 51) could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r = 0.569, p = 0.004; Beclin1/fluoxetine: r = 0.454, p = 0.026; pAkt/amitriptyline: r = −0.416, p = 0.006; pAkt/PAR: r = −0.355, p = 0.021; LC3B-II/PAR: r = 0.453, p = 0.02), as well as by the lymphocytic expression levels of FKBP51 (r = 0.631, p<0.0001), pAkt (r = −0.515, p = 0.003), and Beclin1 (r = 0.521, p = 0.002) at admission. Limitations of the study include the use of male mice only and the relatively low number of patients for protein analyses.

Conclusions

To our knowledge, these findings provide the first evidence for the molecular mechanism of FKBP51 in priming autophagic pathways; this process is linked to the potency of at least some antidepressants. These newly discovered functions of FKBP51 also provide novel predictive markers for treatment outcome, consistent with physiological and potential clinical relevance. Please see later in the article for the Editors'' Summary     

Clinical Relevance of Plasma Prostaglandin F2α Metabolite Concentrations in Patients with Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology with few current treatment options. Recently, we determined an important role of prostaglandin F_2α (PGF_2α) in pulmonary fibrosis by using a bleomycin-induced pulmonary fibrosis model and found an abundance of PGF_2α in bronchoalveolar lavage fluid of IPF patients. We investigated the role of PGF_2α in human IPF by assessing plasma concentrations of 15-keto-dihydro PGF_2α, a stable metabolite of PGF_2α.

Methods

We measured plasma concentrations of 15-keto-dihydro PGF_2α in 91 IPF patients and compared these values with those of controls (n = 25). We further investigated the relationships of plasma 15-keto-dihydro PGF_2α concentrations with disease severity and mortality.

Results

Plasma concentrations of 15-keto-dihydro PGF_2α were significantly higher in IPF patients than controls (p<0.001). Plasma concentrations of this metabolite were significantly correlated with forced expiratory volume in 1 second (Rs [correlation coefficient] = −0.34, p = 0.004), forced vital capacity (Rs = −0.33, p = 0.005), diffusing capacity for carbon monoxide (Rs = −0.36, p = 0.003), the composite physiologic index (Rs = 0.40, p = 0.001), 6-minute walk distance (Rs = −0.24, p = 0.04) and end-exercise oxygen saturation (Rs = −0.25, p = 0.04) when patients with emphysema were excluded. Multivariate analysis using stepwise Cox proportional hazards model showed that a higher composite physiologic index (relative risk = 1.049, p = 0.002) and plasma 15-keto-dihydro PGF_2α concentrations (relative risk = 1.005, p = 0.002) were independently associated with an increased risk of mortality.

Conclusions

We demonstrated significant associations of plasma concentrations of PGF_2α metabolites with disease severity and prognosis, which support a potential pathogenic role for PGF_2α in human IPF.     

Cardiovascular Risk Associated with Interactions among Polymorphisms in Genes from the Renin-Angiotensin,Bradykinin, and Fibrinolytic Systems

Background

Vascular fibrinolytic balance is maintained primarily by interplay of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1). Previous research has shown that polymorphisms in genes from the renin-angiotensin (RA), bradykinin, and fibrinolytic systems affect plasma concentrations of both t-PA and PAI-1 through a set of gene-gene interactions. In the present study, we extend this finding by exploring the effects of polymorphisms in genes from these systems on incident cardiovascular disease, explicitly examining two-way interactions in a large population-based study.

Methodology/Principal Findings

Data from the population-based PREVEND study in Groningen, The Netherlands (n = 8,138) were analyzed. The effects of the polymorphisms and their interactions on cardiovascular events were analyzed via Cox proportional hazards models. There was no association between five of the six polymorphisms singly and risk of cardiovascular disease. There was a significant main effect for the ACE I/D polymorphism for both dominant and additive coding schemes. There were significant interactions between the following polymorphism pairs even after adjustment for known risk factors: ACE I/D & PAI-1 4G/5G (p = 0.012), BDKRB2 C181T & ACE I/D (p = 0.016), BDKRB2 C58T & ACE I/D (p = 0.025), BDKRB2 exon 1 I/D & AT1R A1166C (p = 0.017), and BDKRB2 C58T & AT1R A1166C (p = 0.015).

Conclusions/Significance

This study suggests possible interactions between genes from the RA, bradykinin, and fibrinolytic systems on the risk of cardiovascular disease, extending previous research that has demonstrated that interactions among genes from these systems influence plasma concentrations of both t-PA and PAI-1. Further explorations of these interactions are needed.     

Association of CCR2-CCR5 Haplotypes and CCL3L1 Copy Number with Kawasaki Disease,Coronary Artery Lesions,and IVIG Responses in Japanese Children

Background

The etiology of Kawasaki Disease (KD) is enigmatic, although an infectious cause is suspected. Polymorphisms in CC chemokine receptor 5 (CCR5) and/or its potent ligand CCL3L1 influence KD susceptibility in US, European and Korean populations. However, the influence of these variations on KD susceptibility, coronary artery lesions (CAL) and response to intravenous immunoglobulin (IVIG) in Japanese children, who have the highest incidence of KD, is unknown.

Methodology/Principal Findings

We used unconditional logistic regression analyses to determine the associations of the copy number of the CCL3L1 gene-containing duplication and CCR2-CCR5 haplotypes in 133 Japanese KD cases [33 with CAL and 25 with resistance to IVIG] and 312 Japanese controls without a history of KD. We observed that the deviation from the population average of four CCL3L1 copies (i.e., < or > four copies) was associated with an increased risk of KD and IVIG resistance (adjusted odds ratio (OR)  = 2.25, p = 0.004 and OR = 6.26, p = 0.089, respectively). Heterozygosity for the CCR5 HHF*2 haplotype was associated with a reduced risk of both IVIG resistance (OR = 0.21, p = 0.026) and CAL development (OR = 0.44, p = 0.071).

Conclusions/Significance

The CCL3L1-CCR5 axis may play an important role in KD pathogenesis. In addition to clinical and laboratory parameters, genetic markers may also predict risk of CAL and resistance to IVIG.     

Synthesis,Crystal Structure and Thermal Decomposition of the New Cadmium Selenite Chloride,Cd4(SeO3)2OCl2

A synthetic study in the Cd-Se-O-Cl system led to formation of the new oxochloride compound Cd₄(SeO₃)₂OCl₂ via solid state reactions. The compound crystallizes in the orthorhombic space group Fmmm with cell parameters a = 7.3610(3) Å, b = 15.4936(2) Å, c = 17.5603(3) Å, Z = 8, S = 0.969, F(000) = 2800, R = 0.0185, R_w = 0.0384. Single crystal X-ray data were collected at 293 K. The crystal structure can be considered as layered and the building units are distorted [Cd(1)O₆] octahedra, distorted [Cd(2)O₈] cubes, irregular [Cd(3)O₄Cl₂] polyhedra and SeO₃E trigonal pyramids. There are two crystallographically unique Cl atoms that both are half occupied. Thermogravimetric studies show that the compound starts to decompose at 500°C. The crystal structure of the new compound is closely related to the previously described compound Cd₄(SeO₃)₂Cl₄(H₂O).     

Extracellular Matrix Biomarker,Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

Background

Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS).

Methods

In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo.

Results

During treatment, fibulin-1 became more closely associated with NT-proBNP (β_year0 = 0.10, p = 0.08, β_year1 = 0.16, p = 0.005, β_year4 = 0.22, p<0.001) and suPAR (β_year0 = 0.05, p = 0.34, β_year1 = 0.16, p = 0.006, β_year4 = 0.13, p = 0.03) at the expense of the association to aortic valve area index (AVAI) (β_year0 = −0.14, p = 0.005, β_year1 = −0.08, p = 0.11, β_year4 = −0.06, p = 0.22) independently of age, gender, creatinine, and serum aspartate aminotransferase (Adj.R_year0 ² = 0.19, Adj.R_year1 ² = 0.22, Adj.R_year4 ² = 0.27). Fibulin-1 was unrelated to aortic regurgitation, left ventricular mass, and ejection fraction. In patients with baseline AVAI<0.58 cm²/m² (median value), fibulin-1 was more closely associated to NT-proBNP (β_year0 = 0.25, β_year1 = 0.21, β_year4 = 0.22, all p<0.01), and suPAR (β_year0 = 0.09, p = 0.26, β_year1 = 0.23, β_year4 = 0.21, both p<0.01) independently of age, gender, AST and treatment allocation.

Conclusions

Increased levels of fibulin-1 were independently associated with higher levels of suPAR and NT-proBNP especially in patients with lower AVAI, suggesting that fibulin-1 may be an early marker of AS as well as cardiac fibrosis secondarily to elevated left ventricular hemodynamic load.