Improved measurement of LINE-1 sequence methylation for cancer detection

BackgroundMethylation of long interspersed nuclear element-1 (LINE-1) sequences varies among normal cells and it is often decreased in cancer genomes and white blood cells (WBC) of cancer patients. Current measurement techniques of genome-wide level are inadequate because LINE-1 methylation is distinctive at each locus. Here, we improved the detection of cancer by combining information of LINE-1 methylation pattern and level.MethodsCombined bisulfite restriction analysis (COBRA) of LINE-1, COBRA LINE-1, was used to test cancer cell lines, two oral rinse cohorts, and WBC from normal and cancer patients. COBRA LINE-1 separated LINE-1 sequences into 4 products depending on the methylation statuses of 2 CpG dinucleotides, as follows: 2 unmethylated CpGs (^uC^uC), partial methylation (^mC^uC), 1 methylated CpG (^mC), and 1 unmethylated CpG (^uC).ResultsThe association between ^mC^uC and ^uC^uC was directly correlated in normal cells (r = 0.4895, p = 0.0009) but inversely correlated in cancer (r = ? 0.8979, p = 0.0002). Oral rinse AUC values of ^uC^uC were 0.763 and 0.926 and methylation levels were 0.707 and 0.621, respectively. ^uC^uC, but not overall methylation level, differentiated cancer WBC from normal (p = 0.0082 and p = 0.4830, respectively).ConclusionLINE-1 partial methylation represents hypomethylation in normal cells but hypermethylation in cancer cells. This information improves LINE-1 methylation detection in cancer.     

Early immunologic responses to trauma in the emergency department patients with major injuries

BackgroundA traumatic insult initiates an inflammatory cascade, which is a contributor to cell damage and could be a marker of injury severity.ObjectiveTo compare the initial and 4-h post-injury lymphocyte subsets and cytokine levels between patients with minor and major injury.MethodsProspective, cross-sectional study of trauma patients in an urban level I trauma center. Inclusion criteria: Adult patients with significant mechanism of injury requiring admission. Variables: cell counts (B-cells, Natural Killer cells, monocytes; and CD4 and CD8 T lymphocytes) and cytokines (IL-1, IL-5, IL-6, IL-10, and TNFα). We divided subjects into two groups (major and minor injury). We defined major injury as an injury severity score ≥15, or drop in hematocrit ≥10 points or blood transfusion requirement. Statistical Analysis: Univariate analysis was performed using each inflammatory marker, and multivariate logistic regression analysis was performed to identify the inflammatory markers associated with major injury.Results79 patients were studied (mean age: 35 ± 17, age range: 13–88, 84% male, 38% penetrating trauma, 96% African-American). 25% of patients (n = 20) experienced major injury. Larger base deficit (?3.6 ± 6.2 vs. ?0.9 ± 4.2) levels were observed in major trauma patients. We found that major injury is associated with a drop in absolute CD4 cell count (but not in the CD8 cells), a rise in absolute B-cell count (but not in the NK-cells or monocytes), and a rise in IL-6 (but not in the IL-1, IL-5, IL-10, TNF-a).ConclusionWe found evidence of a measurable early inflammatory response to trauma, using cytokine levels and lymphocyte subset counts.     

Extracorporeal photopheresis in refractory chronic graft-versus-host disease: the influence on peripheral blood T cell subpopulations. A study by the Hellenic Association of Hematology

Extracorporeal photopheresis (ECP) has been established as an effective treatment modality for patients with chronic extensive graft-versus host disease (GVHD). In the present study, we evaluated the influence of ECP on the numbers of CD4+, CD8+, CD20+, CD56+ cells, and on T-regulatory (Tregs), as well as on the numbers of naïve, central memory (CM), and effector memory (EM) T-cells in patients treated for refractory chronic GVHD. Flow cytometric analysis of peripheral blood lymphocytes was performed for the calculation of the different T-cell subsets. Patients with GVHD had a higher percentage of EM-CD4+ cells in comparison with healthy donors (p = 0.046). The percentages of naïve-CD8+, naïve-CD4+, CM-CD8+, CM-CD4+, EM-CD8+, and Tregs were not different between patients with GVHD and healthy donors. Similarly there was no statistical difference in the percentages of naïve, CM, and EM CD4+ and CD8+ cells before and after 3 months of treatment with ECP. However, in the subset of Tregs a statistically significant increase was observed after 3 months of treatment with ECP (p = 0.015). Responders to ECP had statistically significantly higher absolute numbers of CD4+, and CD8+ cells, in comparison with non-responders. These data further support the concept that ECP does not cause immune-suppression, but should be better considered as an immune-modulating treatment.     

Plasma concentrations but not serum concentrations of brain-derived neurotrophic factor are related to pro-inflammatory cytokines in patients undergoing major abdominal surgery

ObjectivesTo investigate peripheral brain-derived neurotrophic factor (BDNF) concentrations in the perioperative period, their relationship with transforming growth factor-β1 (TGF-β1 tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-6 genetics.Design and methodsProspective, observational study. BDNF, TGF-β1, IL-6 and TNF-α were analysed at baseline (T0), 5 h (T1), 24 h (T2) and 5 days (T3) after surgery, in 21 patients. The IL-6 ? 174 G/C polymorphism was genotyped.ResultsSerum BDNF concentrations decreased (P = 0.048), correlated with TGF-β1 (r = 0.610 at T1, r = 0.493 at T2, r = 0.554 at T3). Plasma BDNF concentrations raised (P = 0.049), correlated with IL-6 and TNF-α at T1 (r = 0.495 and r = 0.441, respectively). BDNF response was predictable from TNF-α and IL-6 concentrations and the IL-6 ? 174 G/C genotype.ConclusionSerum and plasma BDNF concentrations could relate to platelet activation and inflammatory response, respectively. IL-6 genetics played a role in the BDNF acute response.     

Monitoring of urinary messenger RNA levels for the prediction of flare in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is characterized by disease flares and remission. We hypothesize that in clinically quiescent SLE patients, the mRNA level of target genes in the urinary sediment is an early indicator of disease flare.MethodsFrom a cohort of 134 adult SLE patients prospectively followed for 56 weeks, we identified 19 patients with a single disease flare. The mRNA level of eight pre-defined target genes in their urinary sediment before disease flare was compared to 19 matched controls with no disease flare during the same period.ResultsUrinary mRNA level remained static in the control group during the study period. Before disease flare, there was a significant increase in the mRNA level of monocyte chemotactic protein (MCP)-1and forkhead box P3 (FOXP3), and decrease in interleukin (IL)-17 and GATA-3, in the urinary sediment. The mRNA level of FOXP3 in urinary sediment increases 8 weeks prior to a flare, which precedes the corresponding change in serum complement and anti-DNA antibody titer, while that of MCP-1, IL-17, and GATA3 began to change 4 weeks prior to a flare. The same pattern of change in urinary mRNA level was observed in patients with mild-to-moderate or severe flare, and those with renal or non-renal flare. The SLE Disease Activity Index (SLEDAI) score at the time of flare significantly correlated with the change in urinary level of IL-17 (r = ? 0.462, p = 0.046) and GATA-3 (r = ? 0.455, p = 0.05), but not MCP-1 or FOXP3, prior to the flare.ConclusionMonitoring of MCP-1, IL-17, GATA-3 and FOXP3 mRNA level in urinary sediment may provide an early clue for detecting disease flare in SLE patients.     

Salivary zinc finger protein 510 peptide as a novel biomarker for detection of oral squamous cell carcinoma in early stages

BackgroundOral squamous cell carcinoma (OSCC) is one of the most frequent malignancies worldwide. Early diagnosis can mean adequate treatment and increase survival.MethodsThis study uses ClinProt technique to identify salivary biomarkers for early diagnosis of OSCC. A total of 77 salivary samples from both OSCC patients (n = 47) and healthy donors (n = 30) were analyzed with MALDI-TOF MS technology.ResultsSalivary peptides from OSCC patients were separated, using C8-functionalized magnetic beads. Three signals (2918.57 Da, 5592.64 Da, and 4372.66 Da) distinguished OSCC patients from controls. Among them, unique peptide 2918.57 Da, identified as a 24-mer peptide of zinc finger protein 510 (ZNF510), was found in 0% of saliva from healthy individuals, versus 25.0% and 60% from OSCC patients with T1 + T2 and T3 + T4 stages, respectively (P < 0.001). ELISA analysis with rabbit anti-ZNF510 peptide sera shows a starkly higher 24-mer ZNF510 peptide level in saliva from OSCC patients than that in controls (P < 0.001). Also, in immunohistochemical analysis of oral tissues, a significantly higher level of ZNF510 was observed in OSCC tissues than in the OSCC free control tissues. Analysis of areas under receiver-operating characteristic (ROC) curves in OSCC early (T1 + T2) and late stages (T3 + T4) shows greater than 0.95.ConclusionsIdentifying 24-mer ZNF510 peptide as OSCC-related salivary biomarkers via proteomic approach proved useful in adjunct diagnosis for early detection rather than specific diagnosis marker for progression of OSCC patients.     

Serum soluble Fas levels in patients with autoimmune rheumatic diseases

Objective:The role of the serum soluble Fas (sFAS) system is unclear in diagnosis of several autoimmune rheumatic diseases although there are present contradictory reports on the levels of serum sFas. We therefore assessed levels of sFAS in serum of patients with autoimmune rheumatic diseases.Patients and methods:We analyzed sFas levels and their relationship to clinical and laboratory data in patients with systemic lupus erythematosus (SLE, n = 32), rheumatoid arthritis (RA, n = 28), Sjögren's syndrome (SS, n = 20) systemic sclerosis (SSc, n = 21), polymyositis/dermatomyositis (PM/DM, n = 15). Patients with osteoarthritis (OA, n = 20) and healthy volunteers (n = 20) were used as controls. Serum levels of sFAS were determined by ELISA. sFas levels greater than mean (normals) + 2 SD were considered as elevated.Results:The mean sFas values were found higher in RA, PM/DM and OA than in control although no differences were found in SSc and SS patients. The mean sFas levels in SLE patients were lower than healthy controls. Elevated sFas rates in RA, PM/DM and SS were found to be 21.4%, 60%, 10% higher than in healthy controls, respectively. sFas levels in SLE and SSc did not differ from control values. Mean sFas levels did not show significant difference between active and inactive patients in all disease groups except PM/DM, RA and OA. No correlations of sFas with relevant disease subsets, laboratory findings and treatment modalities were found.Conclusions:The findings indicate that the serum sFas molecule may provide a useful additional marker for presence and assessment of disease in patients with RA and PM/DM.     

Saliva S100B in professional sportsmen: High levels at resting conditions and increased after vigorous physical activity

BackgroundNeurological dysfunction is a key medical concern in professional sportsmen (PSM). We investigated whether saliva S100B concentrations in PSM and healthy controls are modified before and after training.MethodsWe conducted a case–control-study in 75 patients (25 PSM vs 50 controls) in which S100B saliva concentrations were expressed as absolute values and percentage of change (%) from samples drawn before (T0) and after (T1) training.ResultsNo differences (P > 0.05) between groups were found regarding clinical, monitoring and laboratory parameters. S100B both in PSM and controls was higher at T1 when compared to T0 (P < 0.01). In PSM, S100B was higher than controls (P < 0.001) at T0 and T1. S100B% at T0–T1 was higher (P < 0.001) in PSM and in controls and between PSM and controls (P < 0.001).ConclusionsIncreased saliva S100B levels in PSM before and after training suggest a paracrine/autocrine protein's role connected to stressing activity, which becomes especially evident in PSMs.     

Detection of Epstein-Barr virus in breast carcinoma in Egyptian women

BackgroundThe association of oncogenic EBV with breast carcinoma (BC) is still in controversy.Aim of workAssess the association of EBV with BC in Egyptian women and find possible relationship between prognostic factors of BC and EBV detection.Subjects and methodsParaffin-embedded sections from 40 female patients with primary invasive BC; ductal (n = 32) and lobular (n = 8) and breast tissues from patients with fibrocystic disease (n = 20) as control were screened for presence of EBV by EBV nuclear antigen-1 (EBNA-1) immunostaining and by PCR for EBV-DNA.Results10 / 40 (25%) of the BC specimens stained positively for EBNA-1; EBNA-1 expression was restricted to a fraction 5%–60% of tumor epithelial cells. EBV-DNA was detected in 8 / 10 of BC specimens positive for EBNA-1. Control specimens were negative by both techniques. 7 / 8 (87.5%) of EBV-DNA positive tumors were associated with > 3 lymph nodes involvement.ConclusionEBV is associated with some invasive BC in Egyptian females and may play a role in their etiology.     

Heritability of genetic variants of resistin gene in patients with coronary artery disease: a family-based study

ObjectiveThe resistin gene (RETN) ?420 C > G and + 299 G > A polymorphism was investigated in a case-control study from forty complex Pakistani families with coronary artery disease (CAD) history. Heritability of the susceptible/variant alleles was investigated from parent–offspring trios in these families.MethodResistin levels were determined from 239 individuals by enzyme-linked immunosorbent assay. Genotyping was performed using polymerase chain reaction and restriction fragment length polymorphism.ResultsElevated resistin levels were observed from CAD cases vs. controls (p < 0.0001). The RETN ? 420 C > G and + 299 G > A polymorphism was more prevalent in cases vs. controls (p < 0.0001). The transmission disequilibrium test revealed a significant association of the ? 420 and + 299 polymorphism with CAD (χ² = 34.4, p < 0.0001 and χ² = 31.6, p < 0.0001, respectively).ConclusionElevated resistin, and the RETN ?420 C > G and + 299 G > A polymorphism may contribute to familial CAD. The ? 420 and + 299 variant alleles are transmitted more frequently from parent to affected offspring. This is the first report on the association of the RETN + 299 G > A polymorphism with CAD.     

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

BackgroundThe objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.MethodsOur method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.ResultsBased on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n = 20) and P664L, (n = 5), and their LDLR activities was measured by this method, which was found to be 55.3 ± 8.9% and 63.9 ± 13.8%, respectively, of that of the control group (n = 15). In comparison, the LDLR activity was 86.1 ± 11.6% (K790X) and 73.3 ± 6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p < 0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4 ± 13.2% (K790X) and 76.9 ± 17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1 ± 9.7% (K790X) and 70.3 ± 26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.ConclusionThese results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.     

Serum brain-derived neurotrophic factor in patients with type 2 diabetes mellitus: Relationship to glucose metabolism and biomarkers of insulin resistance

ObjectivesThe aims of this study were to measure serum levels of brain-derived neurotrophic factor (BDNF) in patients with type 2 diabetes mellitus (T2DM) and to investigate the association of these BDNF levels with biomarkers of glucose metabolism and insulin resistance.Design and methodsWe studied 112 patients with T2DM and 80 age- and gender-matched control subjects.ResultsSerum BDNF levels were significantly lower in patients with T2DM compared to control subjects (15.5 ± 5.2 ng/mL vs. 20.0 ± 7.3 ng/mL, P < 0.01). In patients with T2DM, BDNF levels were significantly higher in females than in males (P < 0.01). In the female patients, BDNF was positively related to immunoreactive insulin (IRI) (ρ = 0.458, P < 0.05) and HOMA-R (ρ = 0.444, P < 0.05). Stepwise multiple regression analysis showed a significant relationship between BDNF and IRI (F = 5.294, P < 0.05) in female patients with diabetes.ConclusionsThese findings suggest that BDNF may contribute to glucose metabolism.     

Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease

BackgroundCrohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB).MethodsHuman GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD).ResultsAnti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p < 0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p < 0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmen's rho = 0.493, p < 0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p = 0.0307).ConclusionsAnti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD.     

Anti-centromere antibodies in a large cohort of systemic sclerosis patients: comparison between immunofluorescence, CENP-A and CENP-B ELISA

IntroductionAnti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc) where they are found in 20–40% of patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. During the last few years, to accommodate high throughput diagnostics, a number of laboratories changed from IIF to ELISA assays. The objective of this study was to compare the detection of ACA by IIF to CENP-A and a CENP-B ELISA in a large cohort of SSc patients.MethodsSera collected from SSc patients (n = 834) were tested for ACA by IIF on HEp-2 cells (ImmunoConcepts, Sacramento, CA) and CENP-A and CENP-B ELISA (both Dr. Fooke Laboratorien GmbH, Neuss, Germany). Furthermore, other autoantibodies were determined by QUANTA-Plex^TM SLE 8 profile (INOVA, San Diego, CA), QUANTA Lite® RNA Pol III (INOVA) and PM1-Alpha ELISA (Dr. Fooke).ResultsThe prevalence of ACA was 35.0% by IIF, 41.6% by CENP-A and 57.8% by CENP-B ELISA. When the CENP-A and the CENP-B ELISA results were compared to the IIF, the area under the curve value derived from receiver operating characteristic analysis was 0.98 for both assays. ACA and anti-topoisomerase I antibodies co-occurred in 1.2% (ACA by IIF), in 3.5% (by CENP-A ELISA) and in 7.4% (by CENP-B ELISA). Anti-CENP-A antibodies were negatively associated with anti-Scl-70, anti-RNA Pol III, (both p < 0.0001), anti-U1-RNP (p = 0.008) and anti-PM1-Alpha antibodies (p = 0.0337). The degree of association was dependent on the cut-off value used.ConclusionAlthough we found good agreement between IIF and ELISA for the detection of ACA in SSc, a significant portion of CENP ELISA positive sera did not show the typical ACA staining pattern. Based on these findings, we conclude that an IIF ACA negative result might not rule out the presence of ACA. In addition, new CENP ELISA kits are reliable for the detection of anti-CENP in SSc sera.     

Association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-alpha (+489G/A) polymorphisms with COPD in Croatian population

ObjectiveTo test for possible association of hsp70-2 (+ 1267A/G), hsp70-hom (+ 2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+ 489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population.MethodsGenotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ² test.Resultshsp70-2 (+ 1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR) = 1.50, 95% confidence interval (CI) = 1.00–2.24 and P = 0.061. OR for the GG genotype was 3.47 with CI = 1.26–9.56 and P = 0.04. No association for hsp70-hom (+ 2437T/C), TNF-α (+ 489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms.ConclusionThis study is supporting the association of hsp70-2 (+ 1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+ 2437T/C), TNF-α (+ 489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).     

Lymphocytic enzymes and lipid peroxidation in patients with metabolic syndrome

ObjectivesMetabolic syndrome (MetS) is considered a state of chronic inflammation. This study aimed to ascertain selected parameters of purinergic and cholinergic systems related to glucose metabolism and inflammation, as well as _γ-glutamyltransferase (GGT) and N-acetyl-b-glucosaminidase (NAG) activities and lipoperoxidation in lymphocytes of patients with MetS.Design and methodsThe adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), acetylcholinesterase (AChE), GGT and NAG activities, as well as thiobarbituric acid reactive substances (TBARS) levels were investigated in lymphocytes of patients with MetS (n = 38) and healthy volunteers (n = 41). We also evaluated the insulin levels, anthropometric measurements and routine biochemical analyses.ResultsADA (p < 0.05), DPP-IV and AChE (p < 0.0001) activities were higher in patients with MetS when compared to the control group. Furthermore, we observed correlations between ADA and DPP-IV activities (p = 0.0002; r = 0.5945), TBARS levels and ADA (p = 0.0021; r = 0.5172) and DPP-IV activities (p = 0.0022; r = 0.5010).ConclusionsOur findings showed that MetS might cause tissue distress that disturbed lymphocytic ADA, DPP-IV and AChE activities in response to inflammatory stimuli. These alterations evidence clinical abnormalities, since these enzymatic systems are able to regulate several aspects of adipose tissue function and inflammatory state of MetS and could be used successfully both for preventing and for halting the progression of MetS.     

Identification of pharmacogenetic predictors of lipid-lowering response to atorvastatin in Chilean subjects with hypercholesterolemia

BackgroundStatins are normally the first-line therapy for hypercholesterolemia (HC); however, the lipid-lowering response shows high interindividual variation. We investigated the effect of four polymorphisms in CYP3A4, CYP3A5 and ABCB1 genes on response to atorvastatin and CYP3A4 activity in Chilean subjects with HC.MethodsA total of 142 hypercholesterolemic individuals underwent atorvastatin therapy (10 mg/day/1 month). Serum lipid levels before and after treatment were measured. Genetic variants in CYP3A4 (? 290A>G, rs2740574), CYP3A5 (6986A>G, rs776746) and ABCB1 (2677G>A/T, rs2032582 and 3435C>T, rs1045642) were analyzed by PCR-RFLP. CYP3A4 enzyme activity in urine samples was assessed through determination of 6β-hydroxycortisol/cortisol free ratio (6βOHC/FC).ResultsAfter 4 weeks of therapy, a significant reduction in total cholesterol (TC) and LDL-c was observed (P < 0.001). The G allele for ? 290A>G polymorphism was related to higher percentage of variation in TC and LDL-c (P < 0.001). Moreover, same allele was associated with higher HDL-c variation (P = 0.017). In addition, CYP3A4 enzyme activity was lower in subjects carrying this polymorphism (P = 0.009). No differences were observed for CYP3A5 and ABCB1 variants.ConclusionOur results suggest that presence of G allele for ? 290A>G polymorphism determines a better response to atorvastatin, being also associated with lower CYP3A4 activity in vivo, causing an increased atorvastatin activity.     

Elevated circulatory MMP-2 and MMP-9 levels and activities in patients with rheumatoid arthritis and systemic lupus erythematosus

ObjectivesMatrix metalloproteinases (MMPs) are suggested to play important roles in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study is to examine the MMPs expressions and activities in Taiwanese RA and SLE patients.Design and methodsLevels and activities of plasma MMP-2 and MMP-9 were investigated by enzyme-linked immunosorbent assay and zymography, respectively.ResultsMMP-2 levels in control subjects, RA and SLE patients were 146.1 ± 34.2, 194.0 ± 24.2 and 208.9 ± 75.9 ng/mL respectively, and for MMP-9 were 51.4 ± 57.1, 567.7 ± 313.1 and 208.7 ± 105.5 ng/mL respectively. Both MMP-2 and MMP-9 levels and activities from all patients were significantly higher than that from control subjects.ConclusionsMMP-2 levels in both patients groups were approximately 1.3–1.4 folds higher than that in control subjects, notably, MMP-9 levels were 11- and 4-folds significantly higher, respectively, in RA and SLE patients. The results which MMP-2 and MMP-9 levels and activities are significantly elevated support the involvement of MMPs proteins in these autoimmune disorders.     

Day-to-day variation of late-night salivary cortisol in healthy voluntaries

ObjectivesThe study examines the components of biological variation of nocturnal salivary cortisol in healthy subjects.Design and methodsEight repetitive measurements were performed in seven subjects during a 25-day time period (study A), and then, for comparison, two salivary specimens were taken during two consecutive days from 20 subjects (study B). Salivary cortisol was measured with the Salimetrics HS-Cortisol assay.ResultsMean salivary cortisol (1.27 nmol/L), analytical variation (CVa = 15.4%), within-subject variation (CVi = 34.1%), between-subject variation (CVg = 35.3%), index of individuality (II = 1.06) and reference change value (RCV = 104%) were obtained for study A. Similar results were obtained from the set of samples of study B.ConclusionThe study results show a medium degree of individuality for salivary cortisol. Both conventional reference values and comparison of serial results may be equally used for clinical interpretation. A change greater of 104% between two successive measurements should be considered significant.     

Mitochondrial depolarisation and oxidative stress in rheumatoid arthritis patients

ObjectivesTo investigate mitochondrial membrane integrity, lipid peroxidation and cytotoxicity in peripheral lymphocytes (PL) from rheumatoid arthritis (RA) patients.Design and methodsSouth African black RA patients (HIV^?) were recruited into the study. Mitochondrial membrane potential (Δψ_m) was analysed in PL using the JC-1 dye distribution assay and flow cytometry. Correlations between Δψ_m and clinical parameters were tested for statistical significance. Cytotoxicity (LDH) and lipid peroxidation (thiobarbituric acid reactive substances (TBARS)) was also determined.ResultsOur findings show significantly elevated levels of cytotoxicity (p = 0.0029) and lipid peroxidation (p = 0.0030) in RA. A significantly higher percentage of circulating PL contained depolarised mitochondria (p = 0.0003) which correlated with disease activity and C-reactive protein levels in patients. Collapse of Δψ_m also negatively correlated to absolute lymphocyte counts (r = ? 0.4041; p = 0.0197).ConclusionThese findings suggest a possible role for mitochondrial membrane alterations in the pathology of RA.