Mark Takahashi

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome

An effective tool for the global analysis of both DNA methylation status and protein–chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20 736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein–chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

The synovial fluid adiponectin-leptin ratio predicts pain with knee osteoarthritis

The relationship between adipokines, such as leptin and adiponectin, and cartilage degeneration is being increasingly recognized. We asked what the relationship is between these hormones and patient-reported knee osteoarthritis (OA) pain. We collected demographic data, Short Form McGill Pain scores, Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scores, and synovial fluid (SF) samples from 60 consecutive patients with severe knee OA at the time of joint replacement surgery. SF samples were analyzed for leptin and adiponectin using specific ELISA. Non-parametric correlations and linear regression modeling were used to identify the relationship between the adipokines and pain levels. The correlations between the individual adipokines and the pain scales were low to moderate and consistently less than that for the corresponding adiponectin/leptin (A/L) ratio. Linear regression modeling showed that the A/L ratio was a significant predictor of a greater level of pain on the MPQ-SF (p = 0.03) but not the WOMAC pain scale (p = 0.77). A greater A/L ratio was associated with less pain with severe knee OA and this metabolic pathway may represent a target for novel therapeutics.

Microarray Analysis of the Infrapatellar Fat Pad in Knee Osteoarthritis: Relationship with Joint Inflammation

Objective. To examine differences in genes involved in fat metabolism, energy homeostasis, adipogenesis, and inflammation between endstage and early-stage knee osteoarthritis (OA) infrapatellar fat pads (IFP). Methods. Twenty-nine endstage and 5 early-stage primary OA IFP were harvested at knee surgery. Total RNA was extracted, labeled, and hybridized to whole-genome expression arrays. Unsupervised analysis of all samples using principal components analysis or 2-way hierarchical clustering showed groupings based on tissue source and disease. Statistical testing identified sets of genes that displayed differences between the 2 fat types. Western blot analysis was performed for protein expression of an identified gene of interest. Results. The 29 IFP demonstrated an elevation in the expression of adipokines such as adiponectin and leptin. A statistically significant increased expression was seen for genes of adipogenesis, such as peroxisome proliferator-activated receptor-γ (PPAR-γ), diacylglycerol acyltransferase 2 (DGAT2), cluster of differentiation (CD36), and thyroid hormone responsive spot (THRSP) in the severe OA fat pads as compared to the controls. A subset of 5 patients in the endstage OA group were consistently similar in gene expression to early OA tissue. Protein expression of PPAR-γ2 was 5.4-fold and PPAR-γ1 was 1.4-fold greater in endstage versus early OA tissue. Conclusion. Endstage OA fat pads demonstrated a significant upregulation of genes for fat metabolism and energy homeostasis and a mixed result for inflammatory cytokines.

Relationship between body habitus and joint leptin levels in a knee osteoarthritis population.

Synovial fluid (SF) leptin has been shown to have an association with cartilage degeneration. Our objective was to examine the relationship between different measures of body habitus and SF leptin levels in an end‐stage knee osteoarthritis (OA) population. Sixty consecutive patients with knee OA were surveyed prior to surgery for demographic data. Body habitus was assessed with the body mass index (BMI), waist circumference (WC), and waist–hip ratio (WHR). SF and serum samples were analyzed for leptin and adiponectin using specific ELISA. Nonparametric correlations and linear regression modeling was used to identify the relationship between the measures of body habitus and SF leptin levels. Females had greater levels of leptin than males in both the serum and SF. Significant correlations were found between SF leptin levels and BMI and WC (R2 0.44 and 0.38, respectively; p < 0.05). Regression modeling showed that female gender and WC were independent predictors of a greater SF leptin level independent of age, BMI, and presence of diabetes (p < 0.05). WC may be a more accurate measure of body habitus than BMI in the relationship between the metabolic effects of adipose tissue and OA.

Inflammatory predictors of ongoing pain 2 years following knee replacement surgery

INTRODUCTION The prevalence of unrelieved pain following total knee arthroplasty (TKA) is substantial. OBJECTIVE We asked if cytokine markers of inflammation in preoperative serum or knee synovial fluid (SF) would predict pain 2 years following TKA. METHODS Demographic data and functional outcomes were recorded at baseline and 2 years with the WOMAC index. Serum and SF tissue samples were collected at the time of surgery. Linear regression modeling was used to determine the relationship between SF/serum inflammatory markers and a lesser improvement in self reported pain at two years follow-up. RESULTS Of our 28 patient cohort, significant correlations between serum and SF levels were found for IL-1β (p<0.002), MIP-1β (p<0.001), adiponectin (p<0.001) and leptin (p<0.001). Adjusted analysis showed that greater SF concentrations of TNF-α, MMP-13 and IL-6 were independent predictors of less pain improvement at two years follow-up (p<0.05). CONCLUSIONS Those patients, having ongoing pain despite no clinical or radiological cause, may have an inflammatory profile characterizing a predisposition to ongoing pain after TKA. LEVEL OF EVIDENCE Prognosis study, Level 2.

Obesity-related Adipokines and Shoulder Osteoarthritis

To the Editor: It is increasingly understood that the association between obesity and osteoarthritis (OA) is partially mediated by a systemic, inflammatory effect of adipokines such as leptin (LEP), adiponectin (ADIPO), and resistin. The association of obesity and OA of non-weight-bearing joints, such as the hand, lends further support to this hypothesis1,2. LEP and resistin have been suggested to have a strong proinflammatory influence3,4, while the role of ADIPO in a knee joint is unclear, as some have shown an antiinflammatory effect5 and others suggest a proinflammatory effect6. Similarly, the ADIPO receptors have been identified in the knee joint only6. No study has examined the epidemiologic relationship between obesity and shoulder OA or identified these obesity hormones in the shoulder joint. The objective of our study was to determine if the adipokines LEP, ADIPO, and resistin are present in the shoulder synovial fluid (SF) of patients with OA and to examine the relationships of these hormones to measures of body habitus. We recruited patients awaiting elective arthroscopic rotator cuff repair or shoulder replacement surgery to participate, between 2009 and 2011. We excluded patients with a history of previous shoulder surgery, previous steroid injection, posttraumatic arthritis, or a history of inflammatory arthropathy. Patients undergoing shoulder replacement surgery for a diagnosis of … Address correspondence to Dr. R. Gandhi, Toronto Western Hospital, East Wing 1-439, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. E-mail: rajiv.gandhi{at}uhn.on.ca

Gene Expression Profiles of the Subcutaneous Fat and Infrapatellar Fatpad in Individuals with Early and Endstage Knee Osteoarthritis: A Cross-sectional Analysis

Objective: Previous research has examined gene expression of the IFP from individuals with early and endstage knee OA. However, there is little understanding of whether the differential expression that was found is a local effect associated with OA disease severity. The objective of this study was to compare gene expression of the SAT and IFP both within individuals with early and late stage knee OA. Methods: Knee SAT and corresponding IFP samples were harvested from twenty-nine patients with endstage and five patients with early stage OA at the time of knee surgery. Total RNA was then extracted, labelled and hybridized to Illumina whole genome expression arrays. Arrays were scanned and intensity of hybridization quantified. Filtered data were analyzed to find significantly different expressed genes between the disease stages and fat types. Results: SAT gene expression demonstrated significantly less differential genes between disease states when compared to the gene expression profiles of IFP tissues between individuals with early and endstage OA. Among those with early stage OA, the SAT and IFP tissues were highly dissimilar at the gene expression level, whereas among those with endstage OA these tissues were comparatively more similar. Conclusions: Our findings suggest that the effects of OA on the IFP are localized, more so with later disease stages, while changes in the SAT are less prominent. It is unknown whether or not the localized effects of IFP may be a unique contributor to knee OA or a result of systemic changes occurring in the body. Further research which includes longitudinal assessments with larger cohorts is warranted.