Relationship between SNPs and expression level for candidate genes in rheumatoid arthritis.

OBJECTIVES: The study of polymorphisms of genes differentially expressed may lead to the identification of putative causal genetic variants in multifactorial diseases such as rheumatoid arthritis (RA). Based on preceding transcriptomic results, we genotyped 10 single nucleotide polymorphisms (SNPs) belonging to six genes (S100A8, RNASE2, PGLYRP1, RUNX3, IL2RB, and LY96) showing the highest fold change (> 1.9) when level of expression was compared between RA patients and controls. These SNPs were then analysed to evaluate their role in RA. METHOD: The relationship between gene expression and genotypes of SNPs was first investigated by Kruskal-Wallis and Mann-Whitney tests in RA patients and controls. The genetic association of these SNPs with RA were then analysed using family-based association tests in trio families. RESULTS: We found that RNASE2 gene expression was related to rs2013109 genotypes in 14 RA patients (p = 0.030). The association study in a discovery sample of 200 French trio families revealed a significant association with RA for one SNP, PGLYRP1-rs2041992 (p = 0.019); this association was stronger in trios where RA patients carried the HLA-DRB1 shared epitope (SE) (p = 0.003). However, this association was not found in a replication sample of 240 European trio families (p = 0.6). CONCLUSIONS: Family-based association tests did not reveal an association between RA and any SNP of the candidate genes tested. However, RNASE2 gene expression was differentially expressed in RA patients considering a sequence polymorphism. This result led us to highlight the potential disease-specific regulation for this candidate gene in RA.

Detection of coca alkaloids in oral fluid from coca leaf (tea) consumers: using solid phase extraction to improve validation parameters and widen the detection window.

Hygrine and cuscohygrine, two coca leaf alkaloids, have been previously proposed as markers to differentiate legal and illegal cocaine consumption. This is a very common problem in some countries of South America, where the consumption of coca leaves has a long tradition. Analytical methods focusing on the assessment of coca leaf alkaloids, such as cuscohygrine, hygrine, tropacocaine and t-cinnamoylcocaine, in oral fluid are virtually non-existent in forensic toxicology laboratories worldwide due to their lack of application. However, the problem of differentiating legal and illegal cocaine use in criminal justice, DUID (drug-impaired driving) and WDT (workplace drug testing) programs is growing. Therefore, researchers are obliged to develop methods to measure coca leaf alkaloids (cuscohygrine, hygrine and t-cinnamoylcocaine) in biological matrices for further validation for routine analyses in forensic toxicology laboratories. This work aims to optimize a previously published separation method by protein precipitation in oral fluid by using solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in multiple reaction monitoring (MRM) mode. The use of SPE allowed the matrix effect and the background to be reduced in the chromatograms due to the obtained cleaner extracts. Consequently, improved detection and quantification limits were reached. Findings showed that the detection windows for coca leaf alkaloids were longer than three hours in real oral fluid samples from volunteers who drank a cup of coca tea. These detection windows are quite higher than those previously obtained when using the method based on separation by protein precipitation.

Preliminary findings on the antimony levels of Quiroga river water in the vicinity of a long-abandoned stibnite mine.

A UV-Vis spectrophotometric method was developed as a preliminary approach to the determination of antimony in water samples from a river that flows very close to an abandoned mining site. The analyte is complexed with ammonium pyrrolidinedithiocarbamate and absorbance of the complex is measured at 291.06 nm. The standard additions method is mandatory in view of the matrix effect observed, and the response is linear at least up to 9.3 microg/mL of antimony. The sensitivity of the method is 2.609 x 10(-2) mL/microg, whereas the limits of detection and quantification are, respectively, 0.2 and 0.6 microg/mL. The repeatability, expressed as mean relative standard deviation of the measurements within the calibration range, is 2.0%, whereas the repeatability of the entire procedure is 0.3%. The mean analytical recovery within the calibration range was 102.6%. The method was successfully applied to river water samples.

Understanding the hematopoietic microenvironment in chronic myeloid leukemia: A concise review.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that results from the BCR-ABL gene-induced transformation of a primitive hematopoietic cell. This disease has been extensively studied, and, as a result, a very effective therapy has been developed: the tyrosine kinase inhibitors. Although, there is a significant knowledge about the intrinsic biology of CML cells, alterations in their bone marrow microenvironment are not yet completely understood. In this concise review, we summarized recent findings on the composition and function of the bone marrow microenvironment in CML, and their importance in the progression of the disease and treatment resistance.

Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA).

BACKGROUND: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. OBJECTIVES: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. METHODS: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon alpha (IFNalpha), tumour necrosis factor alpha (TNFalpha) and interleukins IL1beta, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1beta, IL6 and TNFalpha were measured by Luminex bead array technology. Following preincubation with IFNalpha, IL1beta and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNalpha and subsequent TLR stimulation was measured. RESULTS: Synovial TLR3/7 expression was co-expressed with IFNalpha, IL1beta and IL18, but not with TNFalpha, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1beta or IL18 could be detected. Type I IFNalpha increased TLR3/7 mRNA expression whereas IL1beta and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNalpha enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. CONCLUSION: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

Anti-inflammatory therapy with tumour necrosis factor alpha inhibitors improves high-density lipoprotein cholesterol antioxidative capacity in rheumatoid arthritis patients.

OBJECTIVE: High-density lipoprotein (HDL) antiatherogenic functions seem to be diminished during inflammatory conditions such as rheumatoid arthritis (RA). The aim of this study was to investigate the effects of tumour necrosis factor (TNF) inhibition on the antioxidative capacity of HDL in RA. METHODS: Plasma lipids and paraoxonase (PON-1) activity were investigated in 45 RA patients, before and during 6 months of anti-TNF therapy. In addition, HDL was isolated and tested for its ability to inhibit copper-induced oxidation of low-density lipoprotein in vitro. RESULTS: Plasma HDL concentrations did not change considerably after 6 months of therapy. However, stable increases of PON-1 activities were observed throughout the same period (p<0.03). The increases were more obvious when related to HDL or apolipoprotein AI concentrations. HDL total antioxidative capacity significantly improved 6 months after the initiation of anti-TNF therapy (p = 0.015). The initial improvement of PON-1 activity paralleled a decrease in the inflammatory status, whereas specific TNF blockade was likely to be responsible for the long-term effects. CONCLUSIONS: Anti-TNF therapy with infliximab has beneficial effects on lipids through changes in HDL antioxidative capacity, which might be clinically relevant and contribute to the reported protective effect of anti-TNF on cardiovascular morbidity in RA. This emphasises the importance of HDL antiatherogenic capacity for cardiovascular risk in chronic inflammatory conditions.

Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study.

OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.

Gene expression profiling in rheumatoid arthritis: current concepts and future directions.

Over the last years microarray technologies have generated new perspectives for the high-throughput analysis of biological systems. Nowadays, it is possible to monitor thousands of genes in a single experiment. This molecular profiling technology combined with standardised and validated clinical measurements can allow a more precise characterisation of a patient's phenotype, and may lead to the design of therapeutic protocols and procedures better tailored to an individual patient's needs. In this report we provide an overview of expression profiling studies in rheumatoid arthritis (RA). RA is a chronic inflammatory disease in which both genetic and environmental factors are involved. The precise molecular mechanisms underlying RA are not fully understood. A systematic literature search revealed nine array-based expression profiling studies in patients with RA. Findings from these studies were compared with those of linkage and genome-wide association (GWA) studies. Although we observed many differences in study design, analysis and interpretation of results between the different studies, we extracted two sets of genes: (1) those differentially expressed in more than one study, and (2) genes differentially expressed in at least one of the reviewed studies and present in RA linkage or GWA loci. We suggest that both sets of genes include interesting candidate genes for further study in RA.

The tumour necrosis factor receptor superfamily member 1b 676T>G polymorphism in relation to response to infliximab and adalimumab treatment and disease severity in rheumatoid arthritis.

OBJECTIVE: To assess the effect of a functional polymorphism (676T>G, M196R) in the tumour necrosis factor receptor super family 1b (TNFSF1b) gene on disease activity, radiological joint damage and response to infliximab and adalimumab treatment in patients with rheumatoid arthritis (RA). METHODS: Two cohorts of patients with RA were genotyped for the 676T>G polymorphism (rs1061622) in exon 6 of the TNFSF1b gene by restriction fragment length polymorphism analysis. One cohort (n = 234) included patients from the Dutch Rheumatoid Arthritis Monitoring register with detailed information on their response to anti-TNF therapy (infliximab and adalimumab), the other cohort comprised patients from a long-term observational early inception cohort at our centre (n = 248). RESULTS: The 676T>G polymorphism was not associated with anti-TNF response after 3 or 6 months of treatment. Linear regression analysis showed no significant difference in the progression of radiological joint damage during the first 3 and 6 years of disease between the three genotype groups (TT, TG and GG). Additionally, no difference in mean disease activity between genotypes was seen after 3 and 6 years of disease. CONCLUSION: Despite its demonstrated functionality, the 676T>G polymorphism in the TNFSF1b gene does not have a major role in either the response to anti-TNF therapy or in the disease severity or radiological progression in RA.

[Prognostic factors associated with postoperative complications in liver transplant]. / Factores pronósticos de complicaciones postoperatorias en el transplante hepático.

OBJECTIVES: the postoperative evolution of patients submitted to orthotopic liver transplant (OLT) is frequently associated with the appearance of different types of complications such as renal failure, graft rejection, infections, and neurological disorders. These complications are the most significant causes of early morbidity and mortality in patients undergoing OLT. The purpose of the present study was the identification of factors related to the different postoperative complications after OLT. EXPERIMENTAL DESIGN: a prospective study was carried out. PATIENTS: seventy-eight variables were analyzed in 32 consecutive patients undergoing OLT. The factors independently associated with the appearance of postoperative complications were identified using a stepwise logistic regression analysis. RESULTS: the multivariate analysis showed that malondialdehyde and creatinine pretransplant serum levels were associated with the development of renal dysfunction. The pretransplant levels of haemoglobin and the units of platelets administered during surgery were prognostic factors of infections. Acute graft rejection was predicted by ?-glutamyl transpeptidase and total bilirubin serum levels. The pretransplant sodium and glutaredoxin levels in serum were associated with neurological complications. CONCLUSIONS: we propose these markers for the identification of high-risk patients allowing an early surveillance and/or treatment to improve morbidity and survival in patients submitted to OLT.

Adjuvant chemotherapy for prevention of recurrence of invasive hepatocellular carcinoma after orthotopic liver transplantation.

UNLABELLED: Orthotopic liver transplantation (OLT) is the best treatment for nonresectable hepatocellular carcinoma (HCC), but tumor recurrence reduces long-term and medium-term survival. The effectiveness of adjuvant chemotherapy to prevent tumor recurrence has not been fully established. METHODS: Three hundred eighty-seven consecutive patients, including 43 with HCC superimposed on liver cirrhosis, underwent OLT. Twelve patients with one or more prognostic criteria for HCC recurrence were entered into a prospective prophylaxis protocol with monthly cycles of cisplatin (60 mg/m(2)) and adriamycin (30 mg/m(2)), beginning the fourth week post-OLT for a maximum of seven sessions. RESULTS: The 5-year survival of the non-HCC patients was 65.7% and that of the HCC patients was 60.46% (P = NS). Chemotherapy was reasonably well tolerated, but the 9 patients with hepatitis C- or B-associated cirrhosis showed viral and histological recurrence of the primary disease. A high proportion of patients (7 of 12) developed tumor recurrence during the first year after OLT. Six of these patients died, all but one due to HCC relapse. Five patients remain healthy and tumor free at 58 to 130 months. Post-OLT adjuvant chemotherapy does not avoid tumor recurrence and its fatal consequences but may contribute to prolonged tumor-free survival among a significant proportion of patients with high-risk HCC. However, the uncertain implications on viral recurrence and the lack of control groups do not allow post-OLT chemotherapy to be recommended outside controlled clinical trials, which are clearly warranted.

Treatment of refractory cholestatic pruritus with molecular adsorbent recirculating system (MARS).

UNLABELLED: Pruritus is a common complication of cholestatic liver diseases or liver graft dysfunction. Current medical therapies lack efficacy. The molecular adsorbent recirculating system (MARS) represents an interesting therapeutic option. Our objective was to report our experience in the management of four patients with intractable pruritus with MARS. PATIENTS AND METHODS: The MARS treatment cycle included three consecutive treatments, each of 8 hours duration. The four patients with intractable pruritus who were treated had primary biliary cirrhosis/autoimmune hepatitis overlap syndrome (n = 1), ductopenic allograft rejection (n = 2), or posttransplant cholestatic HCV recurrence (n = 1). Intensity of pruritus was documented 24 hours before as well as 24 hours, 7 and 30 days after MARS therapy, and at the end of follow-up. We measured complete blood cell counts, glucose, BUN, creatinine, sodium, potassium, AST, ALT, GGT, alkaline phosphatase, bilirubin, prothrombin activity, and activated partial thromboplastin time. RESULTS: MARS therapy was well tolerated. Patient 1 experienced temporal relief of pruritus, but needed another MARS cycle because of relapse. Patient 2 experienced partial and temporary relief of pruritus, was listed for retransplantation, and received a liver graft 2 months later. Patient 3 showed a dramatic reduction in the degree of pruritus with MARS. Pruritus in patient 4 decreased promptly with MARS therapy and conversion of immunosuppression to tacrolimus, thereby avoiding retransplantation. CONCLUSION: MARS therapy is a promising, safe therapeutic option to treat refractory pruritus caused by cholestatic liver disorders.

[Utility of routine use of reversion after sedation in outpatient colonoscopy]. / Utilidad en la práctica clínica habitual de la reversión tras sedación en colonoscopia ambulatoria.

INTRODUCTION: Randomized clinical trials have demonstrated the effectiveness of flumazenil in reducing recovery time in the endoscopy unit after conscious sedation with midazolam and meperidine. However, its effectiveness in routine clinical practice has not been proved and therefore its use is debated. AIM: To determine the effectiveness of reversion with flumazenil after conscious sedation with midazolam and pethidine in outpatient colonoscopy and to evaluate its cost-effectiveness. MATERIAL AND METHOD: Two hundred consecutive outpatients scheduled for colonoscopy with conscious sedation with midazolam and pethidine in 2 endoscopy units were prospectively included. According to routine clinical practice in each unit, 100 patients were reverted with flumazenil and 100 were not. Medical and demographic data, indications for endoscopy, doses of the medications used and endoscopic findings were collected. Three weeks after the colonoscopy each patient was interviewed by telephone to determine discomfort and complications associated with the endoscopy and/or with the use of sedation. The cost-effectiveness of the use of flumazenil was analyzed. RESULTS: There were no differences between the two groups in age (52.9 +/- 15.5 vs 52.7 +/- 18.3, p = NS), indications for colonoscopy, findings, percentage of full colonoscopies (93 vs 95%; p = NS) or in the doses of sedatives used. The recovery time in each endoscopy unit was similar (19.2 +/- 11.7 vs 15.5 +/- 10.1 minutes, p = NS). In the reversion group there were fewer patients with prolonged stays, defined as those > 20 minutes (23 vs 11%, p < 0.001). The number of patients who remembered discomfort during the colonoscopy was significantly greater in the reversion group (23 vs 13%; p < 0.05). There were no differences in the number of patients who reported discomfort in the days following the endoscopy. The best cost-effectiveness ratio was obtained for a mean stay of 25 min. CONCLUSION: Routine use of reversion does not decrease the mean stay in the endoscopy unit but does decrease the number of prolonged stays. Unpleasant memories of the colonoscopy were more frequent in reverted patients. The effectiveness of the routine use of reversion with flumazenil after conscious sedation with midazolam and pethidine depends on the ratio between the number of daily colonoscopies and the capacity of the recovery room.

Effects of antirheumatic agents on cytokines.

A review of the literature concerning the effects of traditional antirheumatic drugs on cytokines and the cytokine and anticytokine approaches already used in the therapy of rheumatoid arthritis (RA) is presented. Many antirheumatic drugs are capable of cytokine modulation in vitro. Corticosteroids inhibit the transcription of a broad spectrum of genes including those encoding monocyte, T cell-derived cytokines and several hemopoietic growth factors, whereas drugs such as cyclosporin A and D-penicillamine interfere with T cell activation more specifically by suppressing interleukin 2 (IL-2) production. The in vivo effects of drug therapy on cytokines in RA patients are less well established. Gold compounds reduce circulating IL-6 levels and the expression of monocyte-derived cytokines, such as IL-1, tumor necrosis factor (TNF), and IL-6, in the rheumatoid synovium. Decreases in circulating IL-6, soluble IL-2 (sIL-2R), and TNF receptors and in synovial fluid IL-1 levels have been reported with methotrexate. Reductions in circulating IL-6 and sIL-2R concentrations have also been observed with cyclosporin and corticosteroids, whereas azathioprine reduces IL-6 but not sIL-2R. Studies on sulfasalazine are conflicting and the in vivo effects of D-penicillamine and antimalarials have not been studied yet. Interferon gamma therapy is not effective in RA but may prove a useful antifibrotic for systemic sclerosis. Colony stimulating factors improve the granulocytopenia associated with Felty's syndrome or drug toxicities but can induce arthritis flares and should be reserved to treat infectious complications. Promising results are being obtained with selective antagonism of TNF and IL-1 in RA, and combinations of anticytokine strategies with traditional antirheumatic drugs have been already envisaged. These should preferably be based in a broader knowledge of the effects of antirheumatic agents on the cytokine network.

Hepatotoxicity of parenteral gold therapy in rheumatoid arthritis: a case report and review of the literature.

We report a case of severe hepatotoxic reaction during gold therapy for rheumatoid arthritis. The previous literature on this condition is reviewed and the possible mechanisms of gold-induced hepatotoxicity are discussed.

Methotrexate: anti-inflammatory or immunosuppressive?

Low dose methotrexate (MTX) is an effective drug for rheumatoid arthritis, suppressing joint inflammation and even radiographic progression of joint disease. Some disease-specific side effects suggest an immunomodulatory effect of the drug. However, most data currently available suggest a mechanism of action at the level of the non-specific efferent arc of the immuno-inflammatory process rather than on T cells.

Dermatomyositis with subclinical myositis and spontaneous pneumomediastinum with pneumothorax: case report and review of the literature.

Dermatomyositis (DM) is a clinical entity characterized by a distinctive cutaneous rash and inflammatory myopathy. Besides skin and muscle, the disease can also involve other internal organs, especially the lungs. We describe a patient with dermatomyositis and incomplete signs of myositis who developed pneumomediastinum, pneumothorax and massive subcutaneous emphysema. This case illustrates a rare pulmonary complication of DM, and underscores that muscular involvement in this disease is not always reflected in laboratory and/or histological abnormalities.

European genetic study on rheumatoid arthritis: is there a linkage of the interleukin-1 (IL-1), IL-10 or IL-4 genes to RA?

UNLABELLED: The genetic predisposition for rheumatoid arthritis (RA) is only partly explained by the HLA locus and most genetic factors involved in the susceptibility (and/or severity) of the disease await further identification. The first European genome scan in RA families provided suggestive evidence for linkage with a region (3.1/3q13) on chromosome 3, but many other potential RA susceptibility genes have yet to be analysed. AIMS: To perform a linkage analysis with microsatellite markers located in the vicinity of the interleukin-1 (IL-1) gene superfamily, the IL-10 gene and the IL-4 gene cluster which might be considered putative candidate loci for RA. METHODS: 107 Caucasoid European RA sibpairs from 90 nuclear families were genotyped for markers flanking the genes for the IL-1 superfamily, IL-10 and the IL-4 gene cluster. Linkage analysis based on the identity by descent (IBD) in affected siblings was analysed with the program SIBPALNA. Affected sibpairs were stratified according to the identity by state (IBS) for three markers in the HLA region (DRB1 oligotyping, D6S276 and TNFa microsatellites) and to the presence/absence of erosive disease on X-ray examination. RESULTS: Analysis of the whole family set showed an excess of allele sharing for markers of the IL-1 gene cluster (IBD 60%; P = 0.012) but not for IL-10 or IL-4. After stratification, the evidence of linkage to IL-1 was restricted to HLA concordant sibpairs (n = 32; IBD 70%; P = 0.006). Some evidence of linkage to IL-10 was also observed in HLA concordant sibpairs (IBD 66%; P = 0.03) and in sibpairs with erosive disease (n = 61; IBD 62%; P = 0.02). CONCLUSIONS: We found suggestive evidence of linkage of RA to the IL-1 locus. The increased linkage to IL-1 and IL-10 in HLA-identical sibs suggests a possible interaction between these cytokines and the HLA loci. Moreover IL-10 could interact with HLA factors in predisposing to erosive disease. These results need to be tested in additional families for consistency and replication.