The Fidelity of Rheumatoid Arthritis Multivariate Diagnostic Biomarkers Using Discriminant Analysis and Binary Logistic Regression

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that causes multi-articular synovitis. The illness is characterized by worsening inflammatory synovitis, which causes joint swelling and pain. Synovitis erodes articular cartilage and marginal bone, resulting in joint deterioration. This bone injury is expected to be permanent. Cytokines play a prominent role in the etiology of RA and could be useful as early diagnostic biomarkers. This research was carried out at Riyadh’s King Khalid University Hospital (KKUH). Patients were enrolled from the Rheumatology unit. Seventy-eight RA patients were recruited (67 (85.9%) females and 11 (14.1%) males). Patients were selected for participation by convenience sampling. Demographic data were collected, and disease activity measurements at 28 joints were recorded using the disease activity score (DAS-28). Age- and sex-matched controls from the general population were included in the study. A panel of 27 cytokines, chemokines, and growth factors was determined in patient and control sera. Binary logistic regression (BLR) and discriminant analysis (DA) were used to analyze the data. We show that multiple cytokine biomarker profiles successfully distinguished RA patients from healthy controls. IL-17, IL-4, and RANTES were among the most predictive variables and were the only biomarkers incorporated into both BLR and DA predictive models for pooled participants (men and women). In the women-only models, the significant cytokines incorporated in the model were IL-4, IL-17, MIP-1b, and RANTES for the BLR model and IL-4, IL-1Ra, GM-CSF, IL-17, and eotaxin for the DA model. The BLR and DA men-only models contained one cytokine each, eotaxin for BLR and platelet-derived growth factor-bb (PDGF-BB) for DA. We show that BLR has a higher fidelity in identifying RA patients than DA. We also found that the use of gender-specific models marginally improves detection fidelity, indicating a possible benefit in clinical diagnosis. More research is needed to determine whether this conclusion will hold true in various and larger patient populations

Expression and new exon mutations of the human Beta defensins and their association on colon cancer development.

The development of cancer involves genetic predisposition and a variety of environmental exposures. Genome-wide linkage analyses provide evidence for the significant linkage of many diseases to susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Human β-defensins (hBDs) are important molecules of innate immunity. This study was designed to analyze the expression and genetic variations in hBDs (hBD-1, hBD-2, hBD-3 and hBD-4) and their putative association with colon cancer. hBD gene expression and relative protein expression were evaluated by Real-Time polymerase chain reaction (qPCR) and immunohistochemistry, respectively, from 40 normal patients and 40 age-matched patients with colon cancer in Saudi Arabia. In addition, hBD polymorphisms were genotyped by exon sequencing and by promoter methylation. hBD-1, hBD-2, hBD-3 and hBD-4 basal messenger RNA expression was significantly lower in tumor tissues compared with normal tissues. Several insertion mutations were detected in different exons of the analyzed hBDs. However, no methylation in any hBDs promoters was detected because of the limited number of CpG islands in these regions. We demonstrated for the first time a link between hBD expression and colon cancer. This suggests that there is a significant link between innate immunity deregulation through disruption of cationic peptides (hBDs) and the potential development of colon cancer

Differents structures of hBDs: hBD-1 (panel A), hBD2 (Panel B), and hBD3mutant and wild type (panel C and D).

<p>Differents structures of hBDs: hBD-1 (panel A), hBD2 (Panel B), and hBD3mutant and wild type (panel C and D).</p

Clinical data of patients diagnosed with colon cancer via colonoscopy.

<p>Clinical data of patients diagnosed with colon cancer via colonoscopy.</p

Summary of hBDs mutations and their nature/location found in colon cancer tissues.

<p>N = Normal colon tissue</p><p>T = Tumor</p><p>Summary of hBDs mutations and their nature/location found in colon cancer tissues.</p

Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients

<div><p>Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (<i>rs4986790</i>, <i>rs10759932</i>, <i>rs10759931</i> and <i>rs2770150</i>) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 <i>rs10759931</i> polymorphism (OR = 0.086, CI: 0.04–0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the <i>rs2770150</i> is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074–0.48, P = <0.00084). <i>rs10759932</i> and <i>rs4986790</i> appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment.</p></div

Amino acid sequence alignment of four human beta defensins (panel A for hBD1, panel B for hBD2,Panel C for hBD3 and panel D for hBD4) with the amino acid sequences for their corresponding observed mutants.

<p>Mutations are highlighted in red for each hBDs gene.</p

Predicted effect of the mutations affecting hBD-3 on protein structure stability.

<p>^apredicted protein thermal stability change (∆∆G in Kcal/mol) of mutation from CUPSAT program.</p><p>^brelative solvent accessibility of the wild type residue computed from PoP MuSiC program.</p><p>^cpridected protein stability change (∆∆G in Kcal/mol) of mutation from PoP MuSiC program.</p><p>Predicted effect of the mutations affecting hBD-3 on protein structure stability.</p

Human beta defensin (hBD) protein expression in colon cancer tissues.

<p>Tissues were immunostained using specific hBD antibodies (Panel 2A). hBD- positive cells in the tissues were estimated as follows: 0 points, no positive color; 1 point, <20% positive staining; 2 points, 21‑50% positive staining; 3 points, 51–75% positive staining; and 4 points, >75% positive staining. This is presented in Panel B.</p

Human beta defensin (hBD) mRNA and protein expression in colon cancer tissues.

<p>Total cellular RNA freshly extracted from normal and colon cancer tissues was reverse transcribed into cDNA and then used to measure the hBD mRNA expression (Panel1A to 1D).</p