A new treatment for knee osteoarthritis: Clinical evidence for the efficacy of Arthrokinex™ autologous conditioned serum.

OBJECTIVE: The desired therapeutic effect of Arthrokinex™ autologous conditioned serum (ACS) is facilitated by the ability of IL-1-Ra to limit the destructive inflammatory intra-articular (IA) actions of IL-1ß. Previous studies have proven the capacity of Arthrokinex™ (ACS) to induce the anti-inflammatory cytokine, IL-1-Ra. The primary purpose of this retrospective study was to investigate the effect of Arthrokinex™ (ACS) to reduce pain, improve joint function and enhance quality of life in patients with knee osteoarthritis. METHODS: Venous blood from 100 patients with symptomatic knee osteoarthritis (KOA) was conditioned and injected into the affected joint in this treatment protocol. Each patient received a total of six ultrasound-guided IA injections at day 0, 7, 14, 90, 180, and 270 and followed for up to one year. Treatment outcome measures were assessed by three different patient-administered surveys at each visit. Using the Visual Analog Pain Scale (VAS), participants were asked to classify pain in the previous 24 h. The Extra Short Musculoskeletal Functional Assessment (XSMFA-D) survey is a series of 16 questions designed to determine the functionality of the OA-affected joint. Finally, the patient completed a patient global impression of change (PGIC) survey to assess their individual level of satisfaction with the treatment regimen. RESULTS: Compared to baseline, a total of 84% of patients reported better pain control at 6 months with 91% reporting improvement at 12 months. A robust and statistically significant improvement in each XSMFA-D subscale was observed in KOA patients over 12 months. The overall reduction of pain and enhanced joint function was observed within 1 week and sustained 3, 6 and even 12 months after the initial injection. In addition to symptomatic control of OA, 92% of patients reported satisfaction with the treatment regimen 12 months after the initial injection. CONCLUSION: Given the favorable safety profile, reduction in pain and enhanced quality of life experienced by patients enrolled in this joint health program, Arthrokinex™ (ACS) has the potential to offer an alternative, chondroprotective, natural, molecular approach to treating pain and functionality in patients with mild, moderate or severe knee osteoarthritis.

A method to induce Interleukin-1 Receptor Antagonist Protein from autologous whole blood.

OBJECTIVE: Current orthopedic therapies, aimed solely at symptomatic control, are unable to restore the cytokine imbalance that produces the hallmark clinical profile of osteoarthritis. While a myriad of chemical factors in the cytokine network stimulate local joint inflammation and pain, Interleukin 1 (IL-1) is widely recognized as a key offender and a potential therapeutic target. The purpose of this article is to describe a novel, on-site, point of service process (Arthrokinex™) to induce Interleukin 1 Receptor Antagonist Protein (IL-1-Ra or IRAP) from whole blood aimed at inhibiting the destructive intra-articular effects of IL-1. METHODS: 53 patient charts were included in this retrospective chart review study. Venous blood from the selected participants had been harvested and centrifuged to isolate Platelet Rich Plasma and Platelet Poor Plasma. These layers were extracted and incubated for 30 min in a specialized syringe containing medical grade concentrator beads. After centrifuge filtration, the supernatant containing IL-1-Ra was extracted. Anti-inflammatory (IL-1-Ra, IL-10) and pro-inflammatory (TNF α, IL-1 ß) cytokines of baseline whole blood were compared to the conditioned serum following quantification using ELISA. RESULTS: On average, a 32-fold increase (baseline, 550 pg/mL; post conditioning 17,537 pg/mL) in IL-1-Ra concentration was observed after the brief interaction of blood with the concentrator bead surface. IL-1-Ra, if present in concentrations that are 10-100 times higher than IL-1ß, will block the interaction of IL-1ß with cell surface receptors. At these increased concentrations, Arthrokinex™ induced IL-1-Ra joint injections produce an IL-1-Ra to IL-1ß ratio of 999:1. Post conditioning levels of IL-1ß and TNF α were not clinically significant. CONCLUSION: The Arthrokinex™ blood conditioning process has the ability to rapidly induce IL-1-Ra without increasing the pro-inflammatory cytokine profile.

Genome-wide association study identifies a novel locus contributing to type 2 diabetes susceptibility in Sikhs of Punjabi origin from India.

We performed a genome-wide association study (GWAS) and a multistage meta-analysis of type 2 diabetes (T2D) in Punjabi Sikhs from India. Our discovery GWAS in 1,616 individuals (842 case subjects) was followed by in silico replication of the top 513 independent single nucleotide polymorphisms (SNPs) (P < 10⁻³) in Punjabi Sikhs (n = 2,819; 801 case subjects). We further replicated 66 SNPs (P < 10⁻4) through genotyping in a Punjabi Sikh sample (n = 2,894; 1,711 case subjects). On combined meta-analysis in Sikh populations (n = 7,329; 3,354 case subjects), we identified a novel locus in association with T2D at 13q12 represented by a directly genotyped intronic SNP (rs9552911, P = 1.82 × 10⁻8) in the SGCG gene. Next, we undertook in silico replication (stage 2b) of the top 513 signals (P < 10⁻³) in 29,157 non-Sikh South Asians (10,971 case subjects) and de novo genotyping of up to 31 top signals (P < 10⁻4) in 10,817 South Asians (5,157 case subjects) (stage 3b). In combined South Asian meta-analysis, we observed six suggestive associations (P < 10⁻5 to < 10⁻7), including SNPs at HMG1L1/CTCFL, PLXNA4, SCAP, and chr5p11. Further evaluation of 31 top SNPs in 33,707 East Asians (16,746 case subjects) (stage 3c) and 47,117 Europeans (8,130 case subjects) (stage 3d), and joint meta-analysis of 128,127 individuals (44,358 case subjects) from 27 multiethnic studies, did not reveal any additional loci nor was there any evidence of replication for the new variant. Our findings provide new evidence on the presence of a population-specific signal in relation to T2D, which may provide additional insights into T2D pathogenesis.

A replication study of GWAS-derived lipid genes in Asian Indians: the chromosomal region 11q23.3 harbors loci contributing to triglycerides.

Recent genome-wide association scans (GWAS) and meta-analysis studies on European populations have identified many genes previously implicated in lipid regulation. Validation of these loci on different global populations is important in determining their clinical relevance, particularly for development of novel drug targets for treating and preventing diabetic dyslipidemia and coronary artery disease (CAD). In an attempt to replicate GWAS findings on a non-European sample, we examined the role of six of these loci (CELSR2-PSRC1-SORT1 rs599839; CDKN2A-2B rs1333049; BUD13-ZNF259 rs964184; ZNF259 rs12286037; CETP rs3764261; APOE-C1-C4-C2 rs4420638) in our Asian Indian cohort from the Sikh Diabetes Study (SDS) comprising 3,781 individuals (2,902 from Punjab and 879 from the US). Two of the six SNPs examined showed convincing replication in these populations of Asian Indian origin. Our study confirmed a strong association of CETP rs3764261 with high-density lipoprotein cholesterol (HDL-C) (p = 2.03×10(-26)). Our results also showed significant associations of two GWAS SNPs (rs964184 and rs12286037) from BUD13-ZNF259 near the APOA5-A4-C3-A1 genes with triglyceride (TG) levels in this Asian Indian cohort (rs964184: p = 1.74×10(-17); rs12286037: p = 1.58×10(-2)). We further explored 45 SNPs in a ∼195 kb region within the chromosomal region 11q23.3 (encompassing the BUD13-ZNF259, APOA5-A4-C3-A1, and SIK3 genes) in 8,530 Asian Indians from the London Life Sciences Population (LOLIPOP) (UK) and SDS cohorts. Five more SNPs revealed significant associations with TG in both cohorts individually as well as in a joint meta-analysis. However, the strongest signal for TG remained with BUD13-ZNF259 (rs964184: p = 1.06×10(-39)). Future targeted deep sequencing and functional studies should enhance our understanding of the clinical relevance of these genes in dyslipidemia and hypertriglyceridemia (HTG) and, consequently, diabetes and CAD.

Vitamin D Deficiency and Cardio-Metabolic Risk in a North Indian Community with Highly Prevalent Type 2 Diabetes.

OBJECTIVE: The purpose of this investigation was to examine serum vitamin D status in a population of Punjabi ancestry from Northern India with a high prevalence of type 2 diabetes (T2D) and evaluate the effects of 25(OH)D levels on cardio-metabolic traits. RESEARCH DESIGN AND METHODS: We assessed cardiovascular risk factors and 25(OH)D levels in 1,765 participants (887 T2D cases, 878 normoglycemic controls). RESULTS: 76% of individuals were deficient (<50 nmol/L) in vitamin D. A higher percentage of T2D participants(83%) were vitamin D deficient compared to normoglycemic controls (68%)(p<0.0001).The prevalence of vitamin D deficiency increased progressively with body mass index (BMI) categories (p<0.0001): BMI<23 kg/m2, 65%; BMI 23-27.5 kg/m2, 75%; and BMI>27.5 kg/m2, 81%. T2D participants had significantly decreased serum 25(OH)D levels (ß=-0.41, p=2.8 × 10-20). Individuals with low serum 25(OH)D had elevated fasting glucose(ß=-0.18, p=0.022), BMI (ß=-0.71, p=1.4 × 10-7) and systolic blood pressure (ß=-1.68, p=0.006). A positive association of increased 25(OH)D with HOMA-B (ß=0.17, p=8.0×10-6), and C-peptide (ß=0.09, 0.017) was observed. Non-medicated, normoglycemic, non-hypertensive individuals classified as vitamin D deficient (n=289) exhibited a significant increase in fasting glucose (p=0.02) and BMI (p<0.0001) as well as a significant decrease in C-peptide (p<0.0001) and amylin (p<0.0001) compared to vitamin D sufficient controls (n=150). CONCLUSIONS: Vitamin D deficiency appears to be a significant risk factor for T2D severity and associated cardio-metabolic risk. Early intervention may be considered to improve prevention of T2D related cardiovascular complications.

Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis.

Cells of Flavobacterium johnsoniae glide rapidly over surfaces. The mechanism of F. johnsoniae gliding motility is not known. Eight gld genes required for gliding motility have been described. Disruption of any of these genes results in complete loss of gliding motility, deficiency in chitin utilization, and resistance to bacteriophages that infect wild-type cells. Two modified mariner transposons, HimarEm1 and HimarEm2, were constructed to allow the identification of additional motility genes. HimarEm1 and HimarEm2 each transposed in F. johnsoniae, and nonmotile mutants were identified and analyzed. Four novel motility genes, gldK, gldL, gldM, and gldN, were identified. GldK is similar in sequence to the lipoprotein GldJ, which is required for gliding. GldL, GldM, and GldN are not similar in sequence to proteins of known function. Cells with mutations in gldK, gldL, gldM, and gldN were defective in motility and chitin utilization and were resistant to bacteriophages that infect wild-type cells. Introduction of gldA, gldB, gldD, gldFG, gldH, gldI, and gldJ and the region spanning gldK, gldL, gldM, and gldN individually into 50 spontaneous and chemically induced nonmotile mutants restored motility to each of them, suggesting that few additional F. johnsoniae gld genes remain to be identified.

Flavobacterium johnsoniae GldJ is a lipoprotein that is required for gliding motility.

Cells of Flavobacterium johnsoniae glide rapidly over surfaces by an unknown mechanism. Eight genes required for gliding motility have been described. Complementation of the nonmotile mutant UW102-48 identified another gene, gldJ, that is required for gliding. gldJ mutants formed nonspreading colonies, and individual cells were completely nonmotile. Like previously described nonmotile mutants, gldJ mutants were deficient in chitin utilization and were resistant to bacteriophages that infect wild-type cells. Cell fractionation and labeling studies with [(3)H]palmitate indicated that GldJ is a lipoprotein. Mutations in gldA, gldB, gldD, gldF, gldG, gldH, or gldI resulted in normal levels of gldJ transcript but decreased levels of GldJ protein. Expression of truncated GldJ protein in wild-type cells resulted in a severe motility defect. GldJ was found in regular bands that suggest the presence of a helical structure within the cell envelope.

GldI is a lipoprotein that is required for Flavobacterium johnsoniae gliding motility and chitin utilization.

Cells of Flavobacterium johnsoniae glide rapidly over surfaces by an unknown mechanism. Seven genes (gldA, gldB, gldD, gldF, gldG, gldH, and ftsX) that are required for gliding motility have been described. Complementation of the nonmotile mutants UW102-41, UW102-85, and UW102-92 identified another gene, gldI, that is required for gliding motility. gldI mutants formed nonspreading colonies, and individual cells were completely nonmotile. They were also resistant to bacteriophages that infect wild-type cells, and they failed to digest chitin. Introduction of wild-type gldI on a plasmid restored colony spreading, cell motility, phage sensitivity, and the ability to digest chitin to the gldI mutants. gldI encodes a predicted 199-amino-acid protein that localized to the membrane fraction. Labeling studies with [(3)H]palmitate indicated that GldI is a lipoprotein. GldI is similar to peptidyl-prolyl cis/trans-isomerases of the FK506-binding protein family and may be involved in folding cell envelope protein components of the motility machinery.

Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli.

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization.

Cells of Flavobacterium johnsoniae move rapidly over surfaces by gliding motility. The mechanism of this form of motility is not known. Six genes (gldA, gldB, gldD, gldF, gldG, and ftsX) that are required for gliding have been described. Tn4351 mutagenesis was used to identify another gene, gldH, which is required for cell movement. GldH mutants formed nonspreading colonies, and individual cells lacked the cell movements and ability to propel latex spheres along their surfaces that are characteristic of wild-type cells. gldH mutants also failed to digest chitin and were resistant to bacteriophages that infect wild-type cells. Introduction of pMM293, which carries wild-type gldH, restored to the gldH mutants colony spreading, cell motility, the ability to move latex spheres, phage sensitivity, and the ability to digest chitin. gldH encodes a predicted 141-amino-acid protein that localized to the membrane fraction. Labeling studies with [3H]palmitate demonstrated that GldH is a lipoprotein. GldB and GldD, which were previously described, also appear to be lipoproteins. GldH does not exhibit significant amino acid similarity to proteins of known function in the databases. Putative homologs of gldH of unknown function are found in motile (Cytophaga hutchinsonii) and apparently nonmotile (Bacteroides thetaiotaomicron, Bacteroides fragilis, Tannerella forsythensis, Porphyromonas gingivalis, and Prevotella intermedia) members of the Cytophaga-Flavobacterium-Bacteroides group.