Risk HLA class II alleles and amino acid residues in myeloperoxidase-ANCA-associated vasculitis.

A genome-wide association study (GWAS) indicated that myeloperoxidase-ANCA associated vasculitis (AAV) is associated with HLA-DQ. However, susceptibility alleles in these loci have been under-investigated. Here we genotyped 258 Chinese patients with myeloperoxidase-AAV and 597 healthy control individuals at HLA DRB1, DQA1, DQB1 and DPB1, and extracted the encoded amino acid sequences from the IMGT/HLA database. The replication cohort included 97 cases and 107 controls. T cell epitopes of myeloperoxidase were predicted and docked to the HLA molecules. We found DQA1∗0302 (odds ratio 2.34 (95% confidence interval 1.75-3.14)) and DQB1∗0303 (odds ratio 1.89 (1.45-2.48)) were risk alleles for myeloperoxidase-AAV. They are in overt linkage disequilibrium (r2 0.69) and the haplotype DQA1∗0302-DQB1∗0303 presents a significant risk (haplotype score 6.39) as well. Aspartate160 on the DQ α chain (odds ratio 2.06 (1.60-2.67)), encoded by DQA1∗0302, and isoleucine185 on the DQ ß chain (odds ratio 1.73 (1.38-2.18)), encoded by DQB1∗0303, both located in the α2ß2 domains, conferred significant risk for myeloperoxidase-AAV. Homologous modeling showed that DQα∗160D may confer susceptibility to myeloperoxidase-AAV by altering dimerization of the HLA molecules. Thus, more attention should be paid to the roles of amino acids in the α2ß2 domains in addition to the α1ß1 binding groove of HLA class II molecules.

HLA class II alleles differing by a single amino acid associate with clinical phenotype and outcome in patients with primary membranous nephropathy.

Genome-wide associations and HLA genotyping have revealed associations between HLA alleles and susceptibility to primary membranous nephropathy. However, associations with clinical phenotypes and kidney outcome are poorly defined. We previously identified DRB1*1501 and DRB1*0301 as independent risk alleles for primary membranous nephropathy. Here, we investigated HLA associations with demographic characteristics, anti-phospholipase A2 receptor (PLA2R) antibody, treatment response and kidney outcome after a median follow-up of 52 months in 258 patients. DRB1*0301, but not DRB1*1501, was associated with a significantly higher level of PLA2R antibody (odds ratio 1.58, 95% confidence interval 1.13-2.22). Although DRB1*1502, which differs from DRB1*1501 by a single amino acid, was not a risk allele for primary membranous nephropathy (odds ratio 1.01), it was associated with significantly lower estimated glomerular filtration rates both at baseline (1.79, 1.18-2.72) and at last follow-up (1.72, 1.17-2.53), a significantly worse renal outcome by Kaplan-Meier analysis and a significantly higher risk of end-stage renal disease by Cox regression analysis (hazard ratio 4.52, 1.22-16.74). Nevertheless, the absence of remission remained the only independent risk factor for end-stage renal disease by multivariate analysis. DRB1*1502 was also associated with a significantly higher median PLA2R antibody level [161.4 vs. 36.3 U/mL] and showed interaction with DRB1*0301 for this variable. Thus, HLA genes control PLA2R antibody production and primary membranous nephropathy severity and outcome. Additionally, DRB1*1502 behaves like a modifier gene with a strong predictor value when associated with HLA risk alleles. Other modifier genes need further investigations in larger cohorts.

The susceptible HLA class II alleles and their presenting epitope(s) in Goodpasture's disease.

Goodpasture's disease is closely associated with HLA, particularly DRB1*1501. Other susceptible or protective HLA alleles are not clearly elucidated. The presentation models of epitopes by susceptible HLA alleles are also unclear. We genotyped 140 Chinese patients and 599 controls for four-digit HLA II genes, and extracted the encoding sequences from the IMGT/HLA database. T-cell epitopes of α3(IV)NC1 were predicted and the structures of DR molecule-peptide-T-cell receptor were constructed. We confirmed DRB1*1501 (OR = 4·6, P = 5·7 × 10-28 ) to be a risk allele for Goodpasture's disease. Arginine at position 13 (ARG13) (OR = 4·0, P = 1·0 × 10-17 ) and proline at position 11 (PRO11) (OR = 4·0, P = 2·0 × 10-17 ) on DRß1, encoded by DRB1*1501, were associated with disease susceptibility. α134-148 (HGWISLWKGFSFIMF) was predicted as a T-cell epitope presented by DRB1*1501. Isoleucine137 , tryptophan140 , glycine142 , phenylalanine143 and phenylalanine145 , were presented in peptide-binding pockets 1, 4, 6, 7 and 9 of DR2b, respectively. ARG13 in pocket 4 interacts with tryptophan140 and forms a hydrogen bond. In conclusion, we propose a mechanism for DRB1*1501 susceptibility for Goodpasture's disease through encoding ARG13 and PRO11 on MHC-DRß1 chain and presenting T-cell epitope, α134-148 , with five critical residues.

MHC Class II Risk Alleles and Amino Acid Residues in Idiopathic Membranous Nephropathy.

Epitopes of phospholipase A2 receptor (PLA2R), the target antigen in idiopathic membranous nephropathy (iMN), must be presented by the HLA-encoded MHC class II molecules to stimulate autoantibody production. A genome-wide association study identified risk alleles at HLA and PLA2R loci, with the top variant rs2187668 within HLA-DQA1 showing a risk effect greater than that of the top variant rs4664308 within PLA2R1. How the HLA risk alleles affect epitope presentation by MHC class II molecules in iMN is unknown. Here, we genotyped 261 patients with iMN and 599 healthy controls at the HLA-DRB1, HLA-DQA1, HLA-DQB1, and HLA-DPB1 loci with four-digit resolution and extracted the encoded amino acid sequences from the IMGT/HLA database. We predicted T cell epitopes of PLA2R and constructed MHC-DR molecule-PLA2R peptide-T cell receptor structures using Modeler. We identified DRB1*1501 (odds ratio, 4.65; 95% confidence interval [95% CI], 3.39 to 6.41; P<0.001) and DRB1*0301 (odds ratio, 3.96; 95% CI, 2.61 to 6.05; P<0.001) as independent risk alleles for iMN and associated with circulating anti-PLA2R antibodies. Strong gene-gene interaction was noted between rs4664308(AA) and HLA-DRB1*1501/DRB1*0301. Amino acid positions 13 (P<0.001) and 71 (P<0.001) in the MHC-DRß1 chain independently associated with iMN. Structural models showed that arginine13 and alanine71, encoded by DRB1*1501, and lysine71, encoded by DRB1*0301, facilitate interactions with T cell epitopes of PLA2R. In conclusion, we identified two risk alleles of HLA class II genes and three amino acid residues on positions 13 and 71 of the MHC-DRß1 chain that may confer susceptibility to iMN by presenting T cell epitopes on PLA2R.

Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.

Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking.

[Pretreatment with cyclosporin A nanoparticles emulsion protects apoptosis of swine adipose tissue-derived stem cells].

OBJECTIVE: To investigate the effect of cyclosporine A-nanoparticles emulsion (CsA-NP) on protecting apoptosis of swine adipose tissue-derived stem cells (ASC ) and related mechanisms. METHODS: ASC were randomized to six groups: control group,single H2O2 group,CsA or CsA-NP 0.1 mg/ml+H2O2 group,CsA or CsA-NP 1.0 mg/ml+H2O2 group, CsA or CsA-NP 5.0 mg/ml+H2O2 group,CsA or CsA-NP 10.0 mg/ml+H2O2 group. ASC apoptosis was induced by hydrogen peroxide (H2O2100 µmol/L) in vitro. The morphology of apoptotic cells was observed and the number of apoptotic cells was measured. Apoptosis of ASC was detected by flow cytometry using an apoptosis kit. Cell activity was determined by CCK-8 assay. Caspase-3 activity was detected by applying a caspase-3 assay kit. Expression of cytochrome C was investigated by Western blot. RESULTS: H2O2 induced ASC apoptosis was evidenced by morphological and biochemical changes,which could be significantly reduced by pre-treatment with CsA or CsA-NP at concentration of 0.1-10.0 mg/ml, and the best effect was observed at concentration of 5 mg/ml (apoptosis rate: CsA: 10.6% ± 2.8% vs. 25.2% ± 3.8%; CsA-NP: 6.2% ± 2.6% vs. 25.2% ± 3.6% in control group, all P < 0.01). The cell activity was significantly higher in CsA or CsA-NP pre-treated ASC at concentration of 0.1-10.0 mg/ml than in H2O2 group (P < 0.01). Pre-treatment with CsA or CsA-NP (0.1-10.0 mg/ml) significantly down -regulated caspase-3 activity. Furthermore, CsA or CsA-NP (5 mg/ml) completely inhibited the H2O2-induced release of cytochrome C. CONCLUSIONS: These results suggest that CsA-NP and CsA could protect the oxidative stress-induced ASC apoptosis through decreasing the activation of caspase-3 and inhibiting the release of cytochrome C.

[Efficacy of cyclosporine A-nanoparticles emulsion combined with stem cell transplantation therapy for acute myocardial infarction].

OBJECTIVE: To evaluate the efficacy of cyclosporine A-nanoparticles emulsion (CsA-NP) combined with adipose tissue-derived stem cells (ASCs)transplantation therapy for acute myocardial infarction (AMI) in a miniswine model. METHODS: CsA-NP emulsion was prepared by the high-pressure homogenization method. Models were performed by coronary angioplasty for percutaneous balloon occlusion of left anterior descending artery (LAD). A total of 17 miniswines survived after AMI were divided into four groups: control group (n=5), CsA-NP group (n=4), ASCs group (n=4), and CsA-NP+ASCs group (n=4). ASCs or saline were delivered by intracoronary injection one week after AMI.Before cell transplantation and 8 weeks after cell transplantation, delayed-enhanced magnetic resonance imaging (DE-MRI) was performed to evaluate cardiac function and viability. The infarcted myocardium and implanted cells were histologically studied. RESULTS: Eight weeks after treatment, the left ventricular ejection fraction (LVEF)significantly increased in the CsA-NP+ASCs group when compared with the ASCs group [(53.6 ± 2.4)% vs. (48.3 ± 1.8)%, P<0.05]; meanwhile, the infarct size significantly decreased [(6.2 ± 1.7)cm(3) vs.(7.5 ± 0.6) cm(3), P<0.05] and the thickness of the ventricular wall significantly increased (P<0.05). Histology showed that the number of surviving cells increased nearly by three times in the CsA-NP+ASCs group, and the expressions of the cardiomyocyte specific markers (cTnT and α-actin) were detected. Histological samples also showed that CsA-NP+ASCs group reduced fibrotic tissue, and down-regulated the activation of Caspase-3. CONCLUSION: The CsA-NP+ASCs combination therapy can enhance the viability of ASCs by improving LVEF and preventing LV expansion, which may be explained that CsA-NP has the anti-apoptotic effect and can promote the survivals and proliferation of ASCs.

Secular trends in the etiology and comorbidity of hospitalized patients with congestive heart failure: A single-center retrospective study.

OBJECTIVE: To assess the secular trends in the etiology and comorbidity of patients hospitalized with congestive heart failure (CHF). METHODS: Data of 7,319 patients (mean age 59.6 years, 62.1% male) with a primary discharge diagnosis of CHF, hospitalized from January 1, 1993 to December 31, 2007 at the Chinese People's Liberation Army (PLA) General Hospital were extracted and analyzed. These patients were divided into three groups according to hospitalization period: 1993-1997 (n = 1623), 1998-2002 (n = 2444), and 2003-2007 (n = 3252). The etiological characteristics and comorbidities were assessed. RESULTS: Over the study period, the proportion of patients with ischemic heart disease (IHD) increased from 37.2% during the period 1993-1997 to 46.8% during the period 2003-2007, while that with valvular heart disease (VHD) decreased from 35.2% during the period 1993-1997 to 16.6% during the period 2003-2007 (both P < 0.05). Atrial fibrillation (AF) was the most common comorbidity of heart failure (23.2%, 23.0% and 20.6%, respectively, in the three periods). Compared to that of the period of 1993-1997 with that of, the proportion of patients with myocardial infarction, pneumonia, renal function impairment and hepatic cirrhosis of the period of 2003-2007 increased significantly (P < 0.05) and the proportion of patients with chronic obstructive pulmonary disease and atrial fibrillation decreased significantly (P < 0.05). CONCLUSIONS: This study implies that IHD has became a more common etiology of CHF, while VHD has deceased as an etiology of CHF in Chinese patients during the last two decades.

[Fifteen-year evolving trends of etiology and prognosis in hospitalized patients with heart failure].

OBJECTIVE: To investigate the etiological and prognostic changes of hospitalized patients with chronic heart failure. METHODS: This retrospective study analyzed 7319 hospitalized patients (male 62.07%) with validated primary discharge diagnosis of chronic heart failure in Chinese PLA General Hospital in Beijing from January 1, 1993 to December 31, 2007. Etiological characteristics, comorbidities and 30-day hospitalized mortality in the following three periods: 1993 - 1997 (n = 1623), 1998 - 2002 (n = 2444), and 2003 - 2007 (n = 3252) were compared. RESULTS: (1) The patient age increased [(56.0 ± 17.5) years, (57.8 ± 17.6) years and (62.7 ± 15.5) years, P < 0.01] and hospital stay time decreased [(31.3 ± 17.4) days, (22.7 ± 14.1) days and (20.1 ± 15.2) days, P < 0.01] from 1993 to 2007. (2) The common causes of heart failure were coronary heart disease, hypertension, rheumatic valvular heart disease and diabetes mellitus. From 1993 - 1998 to 2003 - 2007, the proportion of patients with coronary heart disease, hypertension and diabetes mellitus rose from 37.2%, 23.3% and 12.3% to 46.8%, 46.7% and 21.1%, respectively (all P < 0.05). Meanwhile the proportion of patients with rheumatic valvular heart disease fell from 35.2% to 16.6% (P < 0.05). (3) The main etiologies and comorbidities were atrial fibrillation, myocardial infarction, pneumonia, chronic obstructive pulmonary disease and renal failure. From 1993 - 1998 to 2003 - 2007, atrial fibrillation was the most common cause of heart failure, and the rate of myocardial infarction, pneumonia and renal failure rose from 11.0%, 8.9% and 5.2% to 14.7%, 14.5% and 9.1%, respectively (all P < 0.05) and the rate of COPD fell from 12.9% to 8.4% (P < 0.05). (4) The 30-day hospitalized mortalities in the three periods were 7.0%, 4.5% and 5.1%, respectively, and the mortalities in the 1998 - 2002 and 2003 - 2007 periods were lower than those of in the 1993 - 1998 period (all P < 0.05). The mortality related to coronary heart disease decreased significantly from 1993 to 2007 (9.3%, 5.0% and 3.8% in the three periods, respectively, P < 0.05). CONCLUSIONS: It is demonstrated that the primary diseases causing heart failure were coronary heart disease, hypertension, diabetes mellitus and rheumatic valvular heart disease, and the former three diseases exhibited a upward trend and the later one exhibited a downward trend. Moreover, the proportion of comorbidities in patients with heart failure increased over the study period. The 30-day hospital mortality exhibited a downward trend and decreased significantly in patients with coronary heart disease or myocardial infarction.

Carbon balance in an alpine steppe in the Qinghai-Tibet plateau.

Carbon fluxes were measured using a static chamber technique in an alpine steppe in the Qinghai-Tibet Plateau from July 2000 to July 2001. It was shown that carbon emissions decreased in autumn and increased in spring of the next year, with higher values in growth seasons than in winters. An exponential correlation (E(carbon)= 0.22(exp(0.09T) + ln(0.31P+ 1)), R(2)= 0.77, P < 0.001) was shown between carbon emissions and environmental factors such as temperature (T) and precipitation (P). Using the daily temperature (T) and total precipitation (R), annual carbon emission from soil to the atmosphere was estimated to be 79.6 g C/m(2), 46% of which was emitted by microbial respiration. Considering an average net primary production of 92.5 g C/m(2) per year within the 2 year experiment, alpine steppes can take up 55.9 g CO(2)-C/m(2) per year. This indicates that alpine steppes are a distinct carbon sink, although this carbon reservoir was quite small.

[Estimation of net primary productivity in middle reaches of Yarlung Zangbo River and its two tributaries].

Based on the MODIS data and field measurement, the net primary production (NPP) in 2000 and 2006 in the middle reaches of Yarlung Zangbo River and its two tributaries in Tibet were estimated by using a mathematic model. The results showed that in study area, the NPP decreased gradually from valley to ridge, which was accorded with the gradients of precipitation and temperature. The annul NPP per unit area was averagely 86.8 g C m(-2) a(-1), and was 2.15 g C m(-2) a(-1) higher in 2006 than in 2000. Farmland ecosystem had the highest annual NPP per unit area (243.1 g C m(-2) a(-1)), while desert ecosystem had the lowest one (36.5 g C m(-2) a(-1)). The average total NPP in the two years was 512.8 x 10(10) g C a(-1), with the value in 2006 being 12.7 x 10(10) g C a(-1) higher than that in 2000. Meadow ecosystem had the highest annual total NPP (194.4 x 10(10) g C a(-1)), while desert ecosystem had the lowest one (30.3 x 10(10) g C a(-1)). In 2000-2006, the NPP value in the areas with strong human disturbance (0-4 km away from road) had a decreasing trend, while that in the areas difficult for human to reach was in adverse.