Single cell transcriptomics in human osteoarthritis synovium and in silico deconvoluted bulk RNA sequencing.

OBJECTIVES: To reveal the heterogeneity of different cell types of osteoarthritis (OA) synovial tissues at a single-cell resolution, and determine by novel methodology whether bulk-RNA-seq data could be deconvoluted to create in silico scRNA-seq data for synovial tissue analyses. METHODS: OA scRNA-seq data (102,077 synoviocytes) were provided by 17 patients undergoing total knee arthroplasty; 9 tissues with matched scRNA-seq and bulk RNA-seq data were used to evaluate six in silico gene deconvolution tools. Predicted and observed cell types and proportions were compared to identify the best deconvolution tool for synovium. RESULTS: We identified seven distinct cell types in OA synovial tissues. Gene deconvolution identified three (of six) platforms as suitable for extrapolating cellular gene expression from bulk RNA-seq data. Using paired scRNA-seq and bulk RNA-seq data, an "arthritis" specific signature matrix was created and validated to have a significantly better predictive performance for synoviocytes than a default signature matrix. Use of the machine learning tool, Cell-type Identification By Estimating Relative Subsets of RNA Transcripts x (CIBERSORTx), to analyze rheumatoid arthritis (RA) and OA bulk RNA-seq data yielded proportions of T cells and fibroblasts that were similar to the gold standard observations from RA and OA scRNA-seq data, respectively. CONCLUSION: This novel study revealed heterogeneity of synovial cell types in OA and the feasibility of gene deconvolution for synovial tissue.

TSG-6 - a double-edged sword for osteoarthritis (OA).

PURPOSE: To explore mechanisms underlying the association of TSG-6 with osteoarthritis (OA) progression. METHODS: TSG-6-mediated heavy chain (HC) transfer (TSG-6 activity) and its association with inflammatory mediators were quantified in knee OA (n=25) synovial fluids (SFs). Paired intact and damaged cartilages from the same individuals (20 tibial and 12 meniscal) were analyzed by qRT-PCR and immunohistochemistry (IHC) for gene and protein expression of TSG-6 and components of Inter-alpha-Inhibitor (IαI) and TSG-6 activity ± spiked in IαI. Primary chondrocyte cultures (n=5) ± IL1ß or TNFα were evaluated for gene expression. The effects of TSG-6 activity on cartilage extracellular matrix (ECM) assembly were explored using quantitative hyaluronan (HA)-aggrecan binding assays. RESULTS: TSG-6 activity was significantly associated (R > 0.683, P < 0.0002) with inflammatory mediators including TIMP-1, A2M, MMP3, VEGF, VCAM-1, ICAM-1 and IL-6. Although TSG-6 protein and mRNA were highly expressed in damaged articular and meniscal cartilage and cytokine-treated chondrocytes, there was little or no cartilage expression of components of the IαI complex (containing HC1). By IHC, TSG-6 was present throughout lesioned cartilage but HC1 only at lesioned surfaces. TSG-6 impaired HA-aggrecan assembly, but TSG-6 mediated HA-HC formation reduced this negative effect. CONCLUSIONS: TSG-6 activity is a global inflammatory biomarker in knee OA SF. IαI, supplied from outside cartilage, only penetrates the cartilage surface, restricting TSG-6 activity (HC transfer) to this region. Therefore, unopposed TSG-6 in intermediate and deep regions of OA cartilage could possibly block matrix assembly, leading to futile synthesis and account for increased risk of OA progression.

Insights into osteoarthritis progression revealed by analyses of both knee tibiofemoral compartments.

OBJECTIVE: To identify disease relevant genes and pathways associated with knee Osteoarthritis (OA) progression in human subjects using medial and lateral compartment dominant OA knee tissue. DESIGN: Gene expression of knee cartilage was comprehensively assessed for three regions of interest from human medial dominant OA (n = 10) and non-OA (n = 6) specimens. Histology and gene expression were compared for the regions with minimal degeneration, moderate degeneration and significant degeneration. Agilent whole-genome microarray was performed and data were analyzed using Agilent GeneSpring GX11.5. Significant differentially regulated genes were further investigated by Ingenuity Pathway Analysis (IPA) to identify functional categories. To confirm their association with disease severity as opposed to site within the knee, 30 differentially expressed genes, identified by microarray, were analyzed by quantitative reverse-transcription polymerase chain reaction on additional medial (n = 16) and lateral (n = 10) compartment dominant knee OA samples. RESULTS: A total of 767 genes were differentially expressed ≥ two-fold (P ≤ 0.05) in lesion compared to relatively intact regions. Analysis of these data by IPA predicted biological functions related to an imbalance of anabolism and catabolism of cartilage matrix components. Up-regulated expression of IL11, POSTN, TNFAIP6, and down-regulated expression of CHRDL2, MATN4, SPOCK3, VIT, PDE3B were significantly associated with OA progression and validated in both medial and lateral compartment dominant OA samples. CONCLUSIONS: Our study provides a strategy for identifying targets whose modification may have the potential to ameliorate pathological alternations and progression of disease in cartilage and to serve as biomarkers for identifying individuals susceptible to progression.

5α-Reductase inhibitors increase acute coronary syndrome risk in patients with benign prostate hyperplasia.

BACKGROUND: This study explored the possible association between the use of two typical 5ARIs (finasteride and dutasteride) and the risk of acute coronary syndrome (ACS) in patients with benign prostate hyperplasia (BPH). METHODS: From the claims data of the Taiwan National Health Insurance (NHI) Taiwan, we identified 1843 ACS cases among BPH patients and randomly selected 7330 controls without ACS, with a similar mean age of 73 years. Multivariate logistic regression analysis estimated the odds ratio (OR) and 95 % confidence interval (CI) for the relationship between the 5ARIs medications and ACS risk. RESULTS: We found that BPH patients who had received treatment with both finasteride and dutasteride were at a higher risk of ACS with an OR of 3.47 (95 % CI 1.05-11.5), compared to patients without 5ARIs treatment. Furthermore, the dosage analysis showed that there were no significant associations between ACS risk and uses of a single drug medication regardless the dosages. The ORs for those who took only dutasteride were 1.07 (95 % CI 0.39-2.99) with low dose and 0.73 (95 % CI 0.38-1.44) with high dose. The ORs for those who took only finasteride were 1.30 (95 % CI 0.89-1.92) with low dose and 0.98 (95 % CI 0.19-5.13) with high dose. CONCLUSION: This population-based nested case-control study suggests that 5ARI use may increase ACS risk among patients with BPH when patients were exposed to both finasteride and dutasteride.

Central sleep apnea in obese children with sleep-disordered breathing.

OBJECTIVES: In contrast to obstructive sleep apnea (OSA), central sleep apnea (CSA) in obese children has received lesser attention. As pediatric CSA is more prevalent than expected and adversely impacts health, this study aims to elucidate the major factors associated with central apnea index (CAI) and compare CSA between obese and non-obese children. METHODS: Retrospective analysis was performed in a tertiary referral medical center. Children with sleep-disordered breathing (SDB) ranging from 2-18 years old were enrolled. All participants completed history taking, otolaryngological examination and overnight polysomnography. CSA was defined as having CAI exceeding 1 h(-1). CAI and the prevalence of CSA were analyzed in children of different age groups, weight statuses and adenotonsillar sizes. RESULTS: A total of 487 cases were included. The prevalence of CSA was 13.3% (65/487). CAI was negatively correlated with age (r=-0.32, P<0.001). Obese children had a significantly lower CAI than that of non-obese ones (0.20 ± 0.36 vs 0.48 ± 0.82 h(-1), P<0.001). Multiple linear regression analysis demonstrated a relationship between CAI, age and obesity as 'CAI=0.883-0.055 × Age -0.22 × (Obesity)'. CONCLUSIONS: In children with SDB, younger ones have a significantly higher CAI than older ones. Additionally, obese children had a lower CAI than non-obese ones.

A nationwide population-based retrospective cohort study: increased risk of acute coronary syndrome in patients with ankylosing spondylitis.

OBJECTIVES: To compare the risk of acute coronary syndrome (ACS) between patients with and without ankylosing spondylitis (AS). METHOD: This retrospective cohort study identified all patients with AS aged ≥ 18 years newly diagnosed from 2000 to 2009, registered in the National Health Insurance Research Database (NHIRD) in Taiwan. The non-AS cohort consisted of fourfold randomly selected control patients free of AS, frequency matched by age, sex, and diagnosis year. The incidence of ACS was determined for both AS and non-AS cohorts. RESULTS: We selected 6262 patients with AS and 25 048 patients without AS. The patients with AS were more prevalent than those without, with co-morbidities of hypertension, diabetes mellitus (DM), hyperlipidaemia, stroke, and peripheral vascular diseases. The overall incidence rate of ACS was higher in the AS cohort than in the non-AS cohort (4.4 vs. 2.9 per 1000 person-years), with an adjusted hazard ratio (aHR) of 1.36 [95% confidence interval (CI) 1.16-1.59]. AS patients with co-morbidities of hypertension, DM, and cancer had an aHR of 7.74 for ACS, compared to those without these co-morbidities. CONCLUSIONS: AS patients are at higher risk of ACS compared with non-AS subjects. Management of CV risk factors should be taken into account for the treatment of patients with AS, especially for patients with co-morbidities of hypertension, DM, and cancer.

Increased level of MSU crystal-bound protein apolipoprotein A-I in acute gouty arthritis.

BACKGROUND: Gout is a common form of inflammatory arthritis that is triggered by the crystallization of monosodium urate (MSU). We investigated the potential proteins that relate to the pathogenesis or the spontaneous resolution of acute gouty arthritis. METHOD: We screened for differentially expressed proteins in the plasma of patients with acute gouty arthritis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) identification. We confirmed these findings in a population study of 209 subjects, and further determined the protein profile of the synovial fluid (SF) from 24 gouty patients during acute attack by liquid chromatography coupled with tandem MS (LC/MS/MS). RESULTS: The highly expressed apolipoprotein A-I (apoA-I) was identified in the plasma of acute gouty patients compared with healthy controls. Moreover, we detected high levels of SF apoA-I in 83.3% of acute gouty patients during attack. From the population study, apoA-I was increasingly associated with normouricaemia, hyperuricaemia, and acute gouty arthritis (ptrend < 0.001), and plasma uric acid (UA) and apoA-I were positively correlated (p = 0.0156). We used a human liver cell model and found that UA enhanced the hepatic apoA-I mRNA expression level (ptrend < 0.01) and apoA-I secretion level (ptrend = 0.002) in a dose-dependent manner. An elevated MSU concentration caused the endogenous apoA-I to deplete gradually. CONCLUSIONS: Based on the role of apoA-I in anti-inflammation, our observational data in acute gout support the hypothesis that apoA-I expression can be induced under the condition of a high concentration of UA and its elevated level may be implicated in the spontaneous resolution of acute gouty arthritis.

Microdeletions/duplications involving TBX1 gene in fetuses with conotruncal heart defects which are negative for 22q11.2 deletion on fluorescence in-situ hybridization.

OBJECTIVES: Conotruncal heart defects (CTD) are associated with del22q11.2 syndrome, which is often diagnosed by fluorescence in-situ hybridization (FISH). However, in those negative for del22q11.2 on FISH, the etiology is usually obscure. We aimed to use high-resolution array comparative genomic hybridization (array CGH) to clarify the underlying genetic causes in these cases. METHODS: In this retrospective study, fetal samples of amniocytes or fibroblasts, taken either for prenatal diagnosis by amniocentesis or for postnatal survey after termination of pregnancy, were obtained from 45 fetuses with CTD and were investigated by cytogenetic analysis including karyotyping and FISH for del22q11.2 syndrome. Eight fetuses with no findings on karyotyping and FISH were investigated further by array CGH, real-time quantitative polymerase chain reaction (qPCR) and Sanger sequencing of TBX1. RESULTS: Array CGH revealed that three of the eight fetuses carried submicroscopic genomic imbalances. Of these, two cases showed similar small microdeletions/duplications in 22q11.2 (one 0.85 kb microdeletion and one 8.51 kb microduplication). The minimal shared region spanned exon 2 of TBX1, a candidate gene responsible for cardiovascular defects in del22q11.2 syndrome. In all eight cases, the array CGH results were confirmed by qPCR, and Sanger sequencing did not detect other molecular pathologies. CONCLUSION: Our findings indicate an association between TBX1 variations and fetal CTD. The results also demonstrate the power of array CGH to further scrutinize the critical gene(s) of del22q11.2 syndrome responsible for heart defects. Array CGH apparently has diagnostic sensitivity superior to that of FISH in fetuses with CTD associated with del22q11.2 (and dup22q11.2) syndrome.

Ultrasound-assisted extraction of phenolic compounds from Phyllanthus emblica L. and evaluation of antioxidant activities.

OBJECTIVE: The objective of this study was to optimize ultrasound-assisted extraction of phenolic compounds from Phyllanthus emblica. METHODS: Extracts obtained by UAE were evaluated for their antioxidant activities. Extraction experiments were carried out with three factors and three levels namely extraction time (varying from 15 to 60 min), ethanol concentration (varying from 50 to 90%) and frequency (varying from 28 to 56 kHz). RESULTS: The results showed that the UAE optimal conditions of extracting total phenol components were as follows: 15 min of extraction time, 60°C of extraction temperature, 70% of ethanol concentration, 56 kHz of ultrasonic frequency and a 1: 50 solid to solvent ratio. Under optimal conditions, the leaching-out rate of phenolic compounds was up to 55.34 mg g(-1) , and the yield of crude extract of P. emblica was up to 56.82%. The results reveal that the yield of phenolic compounds of UAE (56.82%) is higher than that of conventional solvent extraction (16.78%). Furthermore, the antioxidant activities of ethanol extracts obtained by UAE were evaluated in terms of activities of DPPH (1,1'-diphenyl-2-2'-picrylhydrazyl) radical scavenging activity, total antioxidant activity, metal chelating activity, and reducing power. P. emblica extracts obtained by UAE showed high antioxidant activity (26.00, 50.11 and 115.91 µg mL(-1) of IC50 values for DPPH radicals, total antioxidant ability and chelating ability of ferrous ion). CONCLUSION: The result of this study showed that UAE was a suitable method for the extraction of total phenolic compounds. Moreover, the author's main finding in this work is the fact that phenolic compounds from P. emblica show excellent antioxidant activity in multi-test systems.

Direct assessment of articular cartilage and underlying subchondral bone reveals a progressive gene expression change in human osteoarthritic knees.

OBJECTIVE: To evaluate the interaction of articular cartilage (AC) and subchondral bone (SB) through analysis of osteoarthritis (OA)-related genes of site-matched tissue. DESIGN: We developed a novel method for isolating site-matched overlying AC and underlying SB from three and four regions of interest respectively from the human knee tibial plateau (n = 50). For each site, the severity of cartilage changes of OA were assessed histologically, and the severity of bone abnormalities were assessed by microcomputed tomography. An RNA isolation procedure was optimized that yielded high quality RNA from site-matched AC and SB tibial regions. Quantitative polymerase chain reaction (Q-PCR) analysis was performed to evaluate gene expression of 61 OA-associated genes for correlation with cartilage integrity and bone structure parameters. RESULTS: A total of 27 (44%) genes were coordinately up- or down-regulated in both tissues. The expression levels of 19 genes were statistically significantly correlated with the severity of AC degeneration and changes of SB structure; these included: ADAMTS1, ASPN, BMP6, BMPER, CCL2, CCL8, COL5A1, COL6A3, COL7A1, COL16A1, FRZB, GDF10, MMP3, OGN, OMD, POSTN, PTGES, TNFSF11 and WNT1. CONCLUSIONS: These results provide a strategy for identifying targets whose modification may have the potential to ameliorate pathological alterations and progression of disease in both AC and SB simultaneously. In addition, this is the first study, to our knowledge, to overcome the major difficulties related to isolation of high quality RNA from site-matched joint tissues. We expect this method to facilitate advances in our understanding of the coordinated molecular responses of the whole joint organ.

Genome-wide association study of bipolar I disorder in the Han Chinese population.

We report the first genome-wide association study in 1000 bipolar I patients and 1000 controls, with a replication of the top hits in another 409 cases and 1000 controls in the Han Chinese population. Four regions with most strongly associated single-nucleotide polymorphisms (SNPs) were detected, of which three were not found in previous GWA studies in the Caucasian populations. Among them, SNPs close to specificity protein 8 (SP8) and ST8 α-N-acetyl- neuraminide α-2,8-sialyltransferase (ST8SIA2) are associated with Bipolar I, with P-values of 4.87 × 10(-7) (rs2709736) and 6.05 × 10(-6) (rs8040009), respectively. We have also identified SNPs in potassium channel tetramerization domain containing 12 gene (KCTD12) (rs2073831, P=9.74 × 10(-6)) and in CACNB2 (Calcium channel, voltage-dependent, ß-2 subunit) gene (rs11013860, P=5.15 × 10(-5)), One SNP nearby the rs1938526 SNP of ANK3 gene and another SNP nearby the SNP rs11720452 in chromosome 3 reported in previous GWA studies also showed suggestive association in this study (P=6.55 × 10(-5) and P=1.48 × 10(-5), respectively). This may suggest that there are common and population-specific susceptibility genes for bipolar I disorder.

Experimental treatment of bilateral fetal chylothorax using in-utero pleurodesis.

OBJECTIVE: To assess the use and efficacy of in-utero pleurodesis for experimental treatment of bilateral fetal chylothorax. METHODS: This was a study of 78 fetuses with bilateral pleural effusion referred to three tertiary referral centers in Taiwan between 2005 and 2009. Fetuses were karyotyped following amniocentesis and the lymphocyte ratio in the pleural effusion was determined following thoracocentesis. Forty-nine (62.8%) fetuses had a normal karyotype and were recognized to have fetal chylothorax; of these, 45 underwent intrapleural injection of 0.1KE OK-432 per side per treatment. We evaluated clinical (hydrops vs. no hydrops) and genetic (mutations in the reported lymphedema-associated loci: VEGFR3, PTPN11, FOXC2, ITGA9) parameters, as well as treatment outcome. Long-term survival was defined as survival to 1 year of age. RESULTS: The overall long-term survival rate (LSR) was 35.6% (16/45); the LSR for non-hydropic fetuses was 66.7% (12/18) and for hydropic fetuses it was 14.8% (4/27). If we included only fetuses with onset of the condition in the second trimester, excluding those with onset in the third trimester, the LSR decreased to 29.4% (10/34). Notably, 29.6% (8/27) of hydropic fetuses had mutations in three of the four loci examined. CONCLUSIONS: OK-432 pleurodesis appeared to be an experimental alternative to the gold-standard technique of thoracoamniotic shunting in non-hydropic fetal chylothorax. In hydropic fetuses, pleurodesis appeared less effective.

Associations between serum total bilirubin levels and functional dependence in the elderly.

BACKGROUND: Many studies support the role of bilirubin as a cytoprotector in chronic inflammatory diseases, such as stroke and atherosclerosis. AIM: To investigate the relationship between serum total bilirubin levels and functional dependence in older adults. METHODS: Data from the National Health and Nutrition Examination Survey (1999-2002) pertaining to 2235 old adults were analysed. All participants had given a household interview, providing information of five major domains on self-reported functional status (activities of daily living, instrumental activities of daily living, leisure and social activities, lower extremity mobility and general physical activities), had completed serum total bilirubin measurement, and a questionnaire regarding personal health. Poor performance was defined as experiencing difficulty with one or more items in a given domain. Functional dependence was defined as having three or more poor performances in the five major domains. Multiple logistic regression was performed together with quartile-based stratified odds ratio (OR) comparison and trend tests. RESULTS: The OR of functional dependence for each standard deviation increment in the serum total bilirubin level was 0.56 (P = 0.002). After additional adjustment, the inverse association remained essentially unchanged. In quartile-based analysis, participants with higher quartiles of serum total bilirubin tended to have lower ORs of functional dependence. The trends of lower likelihood of functional dependence across increasing quartiles of the serum total bilirubin level were statistically significant (P < 0.05 for all trends). CONCLUSIONS: Higher serum total bilirubin levels were associated with lower likelihood of functional dependence in older adults.

High risk of cross-reactivity between vancomycin and sequential teicoplanin therapy.

WHAT IS KNOWN AND OBJECTIVE: Teicoplanin and vancomycin show similar clinical and bacteriological efficacy in clinical trials. Teicoplanin has been reported to have a lower adverse drug reaction (ADR) rate than vancomycin. Cross-reactivity between these two glycopeptides is controversial. Our aim was to study the cross-reactivity between teicoplanin and vancomycin through an assessment of all the reported ADRs of these drugs in our University hospital. METHODS: Over a period of 2 years, 170 cases of vancomycin therapy, which were closely monitored by doctors and clinical pharmacists, were used to analyse ADRs. Teicoplanin therapy was used as an alternative in cases of vancomycin intolerance. When an ADR related to vancomycin or teicoplanin was suspected, specialists were consulted to confirm if these were true ADR and to determine whether the implicated drug should be stopped. All ADRs for the two glycopeptides were assessed for causality using the Naranjo probability scale. RESULTS AND DISCUSSION: Thirty-eight of 170 patients (22·4%) treated with vancomycin developed ADRs. Twenty-four patients were switched to teicoplanin. However, 14 of those 24 patients (58·3%) developed ADRs. The time of onset of ADRs involving vancomycin was 12·7 ± 10·9 days (range, 1-46 days). The time of onset of sequential teicoplanin-induced ADRs was 11·7 ± 4·7 days (range, 2-20 days). Of the 14 patients with ADRs related to sequential teicoplanin therapy, six showed cross-reactivity between vancomycin and teicoplanin. The incidence of vancomycin-induced neutropenia was 4·7% (8/170), whereas the incidence of teicoplanin-induced neutropenia subsequent to vancomycin intolerance was as high as 33·3% (8/24). Furthermore, 71·4% (10/14) of the teicoplanin-induced ADRs were associated with haematological abnormalities such as neutropenia, thrombocytopenia or leucopenia. WHAT IS NEW AND CONCLUSION: Teicoplanin, used as an alternative in cases of vancomycin intolerance, was associated with a high incidence of ADRs and haematological reactions, most notably neutropenia. This high rate of ADRs suggests cross-reactivity between the two glycopeptides.

Clinical and microbiological characteristics of urine culture-confirmed genitourinary tuberculosis at medical centers in Taiwan from 1995 to 2007.

All patients with urine culture-confirmed genitourinary tuberculosis (GUTB) diagnosed between 1995 and 2007 at two medical centers in northern Taiwan were included in this retrospective study. Genotypes of 48 preserved Mycobacterium tuberculosis (MTB) isolates from these patients were determined by spoligotyping and double repetitive element PCR (DRE-PCR) analysis. Among the 64 patients, 38 (59.4%) were male with a mean ±SD age of 60.3 ± 16.1 years old. The overall mortality rate was 26.2%. Poor prognostic factors included age over 65 years (HR = 4.03; 95%; CI: 1.27-12.76), cardiovascular disease (HR = 5.96; 95% CI: 1.98-17.92), receiving steroids (HR = 10.16; 95% CI: 2.27-45.47), not being treated (HR 4.81; 95% CI 1.12-20.67). Spoligotyping and DRE-PCR of the 48 MTB isolates revealed that 20 (41.7%) belonged to the Beijing family and 40 (83.3%) had a clustering pattern. Identification of a Beijing family isolate was not correlated with drug resistance or mortality. Clustering strains were likely to be resistant to isoniazid (OR = 4.71; 95% CI: 1.10 to 23.53). In this study of patients with urine culture-confirmed GUTB, age and coexisting diseases were independently associated with an unfavorable outcome. The Beijing family was the dominant genotype of GUTB isolates, but did not correlate with drug resistance or outcome.

Skin and soft-tissue infection caused by non-tuberculous mycobacteria in Taiwan, 1997-2008.

The aim of this study was to investigate the clinical, microbiological, and pathological characteristics and the outcomes of skin and soft-tissue infection (SSTI) caused by non-tuberculous mycobacteria (NTM). Medical records of 50 patients with SSTI caused by NTM identified from 2005 to 2008 and 63 patients previously reported in a medical centre from 1997 to 2004 were reviewed. The annual incidence (per 100,000 outpatients and in-patients) ranged from 0·57 in 2005, 0·38 in 2007, to 1·1 in 2008, with an average of 0·62/100,000. From 1997 to 2008, the average incidence was 1·39/100,000 patients. The average annual incidence of SSTI caused by NTM was 0·62/100,000 outpatients and in-patients during 2005 and 2008. Of the total of 113 patients identified during the 12-year period, patients infected with Mycobacterium fortuitum and M. marinum were younger than those infected with M. avium-intracellulare complex (MAC) (36 and 44 years vs. 55 years, P=0·004 and P=0·056, respectively), and were more likely to have previous invasive procedures than those infected with MAC and M. abscessus (81·8% and 72·0% vs. 27·8% and 54·8%, P=0·007), and less likely to have associated immunosuppression (9·1% and 24% vs. 66·7% and 45·2%, P=0·006). Granuloma was more often observed in immunocompetent patients (60·1% vs. 40%, P=0·019), and in M. marinum-infected specimens (78·3%). There were significant differences in the demographic and clinical features of patients with NTM SSTI, including immunosuppression, trauma experience, and depth of tissue infections.

Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen.

The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.

Molecular internalization of a region of myelin basic protein.

The conformation of myelin encephalitogenic or basic protein (BP) was investigated with a double-antibody radioimmunoassay by studying the reaction of BP or its fragments with antibodies produced in two rabbits against peptide 43-88 linked to rabbit serum albumin. Both antisera reacted well with peptide 43-88 but showed little or no reaction with BP. Absorption of these antisera with a BP-immunoadsorbent did not remove the antibody activity against peptide 43-88. Within the region of peptide 43- 88 it was shown that peptides 68-88 and 79-88 gave an equivalent or better reaction than peptide 43-88, whereas peptides 43-67 and 64-73 had very little reactivity. In the BP fragments containing region 43-88, peptide 1-88 showed the best reactivity, peptide 20-166 showed minimal reactivity, while peptide 1-115 showed none. These data document the internal position of at least a portion of peptide 43-88 and all of residues 79-88 in the BP molecule. The much greater reactivity of peptide 1-88 as compared to peptide 1-115 suggests that the region or a portion of the region of BP containing residues 89- 115 participates in the conformational alignment of BP restricting access to peptide 79-88. After absorption with BP, neither of the antisera prepared to peptide 43-88 reacted with PNS myelin in fixed tissue sections but continued to react with CNS myelin in similarly treated sections. The present findings demonstrate the need to consider the role of shielded antigenic determinants in the investigation of antigens or of immune responses.

The chondroprotective effects of ferulic acid on hydrogen peroxide-stimulated chondrocytes: inhibition of hydrogen peroxide-induced pro-inflammatory cytokines and metalloproteinase gene expression at the mRNA level.

OBJECTIVE: The objective of the study is to evaluate the effect of ferulic acid (FA), an antioxidant from the Chinese herb Dong-Gui [Chinese angelica, Angelica sinensis (Oliv.) Diels], on the regulation of various genes in hydrogen peroxide-stimulated porcine chondrocytes at the mRNA level. METHODS: The effect of FA and the effective concentration of FA on porcine chondrocytes was evaluated by the lactate dehydrogenase, WST-1, crystal violet assay, and a chemical luminescence assay. Gene expression in hydrogen peroxide-stimulated chondrocytes either pre- or post-treated with FA was evaluated by real-time PCR. RESULTS: Chondrocytes pre-treated with 40 microM FA decreased the hydrogen peroxide-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and MMP-1 and partially restored SOX9 gene expression. Post-treatment with 40 microM FA also decreased the expression of MMP-1 and MMP-13. CONCLUSION: FA decreased the hydrogen peroxide-induced IL-1beta, TNF-alpha, MMP-1 and MMP-13 and increased SOX9 gene expression. These findings suggest that FA may prove to be important in the treatment of osteoarthritis. Further research is needed.

Clinical and genotypic characteristics of extensively drug-resistant and multidrug-resistant tuberculosis.

The aims of this study were to compare the clinical features of patients with extensively drug-resistant tuberculosis (XDRTB) and multidrug-resistant tuberculosis (MDRTB) and the genotypic characteristics of these Mycobacterium tuberculosis isolates. A total of 90 non-HIV-infected patients having MDRTB (n = 80, not including XDRTB, 88.9%) and XDRTB (n = 10, 11.1%) were identified from 2000 to 2007. Genotypes of the 39 available isolates were evaluated by spoligotyping and the 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) scheme. Patients with XDRTB were more likely to have previous history of TB and cavitary lung lesions than patients with MDRTB (P < 0.05). Among the 39 isolates for spoligotyping analysis, the Beijing genotype was the most common (n = 21, 53.8%). Four (44.4%) isolates of XDRTB and 17 (56.7%) isolates of MDRTB belonged to Beijing family genotypes. There was no significant difference in the anti-tuberculosis drug resistance rates between Beijing and non-Beijing genotype isolates or in the clinical features of infected patients. In conclusion, significant differences in clinical manifestations existed among patients with XDRTB and MDRTB. The clinical features of patients infected with the Beijing genotype and the drug resistance profile of the Beijing genotype isolates were similar to those for the non-Beijing family genotype.