Effects of balneological outpatient treatment on clinical parameters and serum cytokine levels in patients with chronic low back pain: a single-blind randomized controlled trial.

This study aimed to investigate the effects of balneological outpatient treatment (hydrotherapy and peloidotherapy) on clinical status and serum cytokine levels in patients with chronic low back pain (CLBP). Seventy-four patients with CLBP who accepted to participate to the study were randomly divided into two groups. The study group was given ten sessions (in 2 weeks) of hydrotherapy, peloidotherapy, and home exercise, while the control group was given only home exercise. All patients were assessed before and at the end of therapy, at the 1st and 3rd months. The primary outcomes were pain intensity on the visual analog scale (VAS) (VAS-pain, VAS-rest, VAS-exercise) and Oswestry Disability Index (ODI). The secondary outcome measures included patient's and physician's global assessment (VAS-PGA), (VAS-DGA), finger-to-floor distance (FFD), modified Schober test, Short Form-36 (SF-36), and the use of analgesic drug. Venous blood samples were drawn from all patients before/1st day and after therapy/12th day to measure serum interleukin (IL)-6 and IL-10 levels. Significant improvement was observed in the study group in VAS-pain, VAS-rest, VAS-exercise, VAS-PGA, VAS-DGA, ODI, and SF-36 parameters after treatment and improvement maintained for 3 months. In the control group, significant improvement was observed in VAS-pain, VAS-exercise, VAS-PGA, VAS-DGA, and ODI scores on the 12th day and continued for 3 months. Decrease in pain, pain during rest and exercise, modified Schober test, VAS-PGA, VAS-DGA, ODI scores, and the increase in SF-36 pain and general health scores showed superiority in favor of the study group in all evaluations. There was a significant increase in IL-10 values from baseline at the end of treatment in the study group. The use of non-steroidal anti-inflammatory drug (NSAID) was significantly lower in the study group compared with the use of NSAID in the control group in the 3rd month. Balneological outpatient treatment improved clinical status in CLBP patients. Although no significant correlation was clearly determined between IL-10 levels and pain score, this effect might be related to the observed increase in the anti-inflammatory cytokine IL-10 levels that was observed only in the study group.

The treatment effect on peripheral B cell markers in antibody positive myasthenia gravis patients.

B cells play a major role in the pathophysiology of myasthenia gravis (MG) with their ability to produce disease specific, pathogenic antibodies. However, their status during disease development and follow-up stages of the disease in the peripheral blood may need further studies to determine useful markers. In this study, we aimed to detect B cell associated factors concerning immunosuppressive treatment in generalized non-thymomatous MG patients. Although CD19+ B cell distribution did not vary among disease subgroups, expressions of both CD38 and BAFFR were altered on B cells in MG patients under immunosuppressive therapy. Serum levels of BAFF were elevated in untreated MG patients as compared to treated MG patients and healthy controls. B cell activation factors may show profound alterations due to immunosuppression.

Relation of HLA-DRB1 to IgG4 autoantibody and cytokine production in muscle-specific tyrosine kinase myasthenia gravis (MuSK-MG).

A small subset of myasthenia gravis (MG) patients develop autoantibodies against muscle-specific kinase (MuSK), which are predominantly of the immunoglobulin (Ig)G4 isotype. MuSK-MG is strongly associated with HLA-DRB1*14, HLA-DRB1*16 and HLA-DQB1*05. In this study, the possible effects of these HLA associations on MuSK IgG autoantibody or cytokine production were investigated. Samples from 80 MG patients with MuSK antibodies were studied. The disease-associated HLA types were screened in the DNA samples. The IgG1, IgG2, IgG3 and IgG4 titres of the MuSK antibodies and the levels of interleukin (IL)-4, IL-6, IL-17A and IL-10 were measured in the sera. Comparisons were made among the groups with or without HLA-DRB1*14, HLA-DRB1*16 or HLA-DQB1*05. The IgG4 titres of the MuSK antibodies were higher than those of the IgG1, IgG2 and IgG3 isotypes among the whole group of patients. DRB1*14 (+) DRB1*16 (-) patients had higher levels of IgG4 antibodies than those of DRB1*14 (-) DRB1*16 (+) patients. DRB1*14 (+) DRB1*16 (+) patients also had higher levels of IgG4 antibodies than those of DRB1*14 (-) DRB1*16 (+) and DRB1*14 (-) DRB1*16 (-) patients. Higher IL-10 and lower IL-17A levels were measured in DRB1*14 (+) DRB1*16 (-) patients than in DRB1*14 (-) DRB1*16 (-) patients. The higher IgG4 titres of MuSK autoantibodies in patients carrying HLA-DRB1*14 than those in the other patients suggest a role for HLA in the production of the antibodies. The differences in IL-10 and IL-17A support the role of DRB1 in the etiopathogenesis of this autoimmune response.

The effect of interleukin (IL)-21 and CD4+ CD25++ T cells on cytokine production of CD4+ responder T cells in patients with myasthenia gravis.

Impairment of the suppressive function of regulatory T (Treg ) cells has been reported in myasthenia gravis (MG). In this study, cytokine-related mechanisms that may lead to the defect of Treg were investigated in patients with anti-acetylcholine receptor antibody-positive MG (AChR + MG). Proliferation and cytokine production of responder T (Tresp ) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)-21 on suppression was evaluated in vitro in co-culture. IL-21 increased the proliferation of Tresp cells in Tresp /Treg co-cultures. Tresp cells from patients with MG secreted significantly lower levels of IL-2. In patients with MG, IL-2 levels did not change with the addition of Treg to cultures, whereas it decreased significantly in controls. In Tresp /Treg co-cultures, IL-4, IL-6 and IL-10 production increased in the presence of Treg in patients. Interferon (IFN)-γ was decreased, whereas IL-17A was increased in both patient and control groups. IL-21 inhibited the secretion of IL-4 in MG and healthy controls (HC), and IL-17A in HC only. The results demonstrated that IL-21 enhances the proliferation of Tresp cells in the presence of Treg . An effect of IL-21 mainly on Tresp cells through IL-2 is implicated.

Titin antibodies in "seronegative" myasthenia gravis--A new role for an old antigen.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.

MuSK autoantibodies in myasthenia gravis detected by cell based assay--A multinational study.

Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.

Activation of the JAK/STAT pathway in Behcet's disease.

Th1/Th17-type T-cell responses are upregulated in Behcet's disease (BD). However, signaling pathways associated with this aberrant immune response are not clarified. Whole-genome microarray profiling was performed with human U133 (Plus 2.0) chips using messenger RNA of isolated CD14(+) monocytes and CD4(+) T cells from peripheral blood mononucleated cell (PBMC) in patients with BD (n = 9) and healthy controls (HCs) (n = 9). Flow cytometric analysis of unstimulated (US) and stimulated (phytohaemagglutinin) signal transducer and activator of transcription (STAT3) and pSTAT3 expressions of PBMCs were also analyzed (BD and HC, both n = 26). Janus family of kinase (JAK1) was observed to be upregulated in both CD14(+) monocytes (1.95-fold) and CD4(+) T lymphocytes (1.40-fold) of BD patients. Using canonical pathway enrichment analysis, JAK/STAT signaling was identified as activated in both CD14(+) monocytes (P = 9.55E-03) and in CD4(+) lymphocytes (P =8.13E-04) in BD. Interferon signaling was also prominent among upregulated genes in CD14(+) monocytes (P = 5.62E-05). Glucocorticoid receptor signaling and interleukin (IL-6) signaling were among the most enriched pathways in differentially expressed genes in CD14+ monocytes (P = 2.45E-09 and 1.00E-06, respectively). Basal US total STAT3 expression was significantly higher in BD (1.2 vs 3.45, P < 0.05). The JAK1/STAT3 signaling pathway is activated in BD, possibly through the activation of Th1/Th17-type cytokines such as IL-2, interferon (IFN-γ), IL-6, IL-17 and IL-23.

Serum cytokine profiles in Takayasu's arteritis: search for biomarkers.

OBJECTIVES: Assessment of disease activity is one of the major difficulties in patients with Takayasu arteritis (TAK) during follow-up. To date, no biomarker is universally accepted to be a surrogate for active disease in TAK. In this study, we aimed to investigate levels of various pro-and anti-inflammatory molecules including serum granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, IL-10, IL-18 and IL-23 in patients with TAK. METHODS: The study included 51 patients (age: 40.6±12.2 years, F/M: 45/6) with TAK and 42 age- and sex-matched healthy controls (age: 38.1±7.4 years, F/M: 38/4). All patients fulfilled the criteria of the American College of Rheumatology (ACR). TAK patients were evaluated by physician's global assessment (PGA; active/inactive) and ITAS2010 (Indian Takayasu Arteritis Clinical Activity Score) in terms of clinical activity in baseline and follow-up visits. Commercial enzyme linked immuno-sorbent assay (ELISA) kits were used for measurements of serum cytokine levels. RESULTS: At baseline, 21 (41.2%) patients were active according to PGA and 8 (15.7%) according to ITAS2010. Serum IL-6, IL-8 and IL-18 levels were significantly higher in patients with TAK, whereas GM-CSF, IL-10, IL-23 levels were similar to healthy controls. IL-8 significantly decreased in the follow-up, associated with a decrease of clinical activity, whereas IL-23 level significantly increased. When assessed by ITAS2010 active patients had significantly higher IL-18 levels. CONCLUSIONS: We found significantly increased IL-6, IL-8 and IL-18 levels in patients with TAK compared to healthy controls. Only IL-18 level was significantly higher in active patients assessed by ITAS2010. IL-18 was also the only cytokine in our study that correlated with CRP. These findings suggest that cytokines associated with neutrophilic, pro-inflammatory responses such as IL-6, IL-8 and IL-18 can be potential biomarkers for the assessment of disease activity in TAK and warrant further studies in larger series.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

COMT Val 158 Met polymorphism is related with interpersonal problem solving in schizophrenia.

OBJECTIVE: The aim of this study was to investigate associations between COMT Val(158)Met polymorphism, and interpersonal problem solving capacity and cognitive functions in schizophrenia. METHODS: COMT Val(158)Met polymorphism was studied with ARMS-PCR method in 99 outpatients with schizophrenia. Brief Psychiatric Rating Scale was used to assess symptom severity. The Assessment of Interpersonal Problem Solving Skills (AIPSS) was used to evaluate problem solving capacity. Continuous Performance Test (CPT) and Wisconsin Card Sorting Test (WCST), were used to measure cognition. RESULTS: Patients with Met/Met genotype had higher AIPSS subscores for detecting the problem, than those with Val/Val at baseline (p=0.02). Met allele was also found to be related with higher AIPSS-receiving skills (p=0.04). Val allele was found to be related with more commission errors in CPT (p=0.03). There was no relation between Val(158)Met polymorphism and WCST and clinical measurements. CONCLUSION: Our findings suggest that Val allele might be related to poor performance on detecting the interpersonal problems, and attention in schizophrenia.

No association of PTPN22 gene polymorphism with rheumatoid arthritis in Turkey.

Although the association of rheumatoid arthritis (RA) with HLA-DRB1 (shared epitope) is well demonstrated in many ethnic populations, the role of other RA-associated risk loci is not clarified. In this study, the functional single nucleotide polymorphism (SNP) of PTPN22 gene was investigated in Turkey. 167 patients with RA and 177 healthy controls are genotyped by polymerase chain reaction (PCR)-RFLP for the SNP (rs2476601, A/G) of PTPN22 gene. Polymorphic region was amplified by PCR and digested with Xcm I enzyme. Heterozygous genotype (AG) was present in 5.1% (9/177) of the controls and in 6.6% (11/167) of RA group (p = 0.55, OR 1.3, 95% CI 0.53­3.26). There was also no association between any clinical feature, RF positivity and presence of this SNP. In conclusion, the distribution of PTPN22 polymorphism did not reveal any association with RA in Turkey.

PTPN22 gene polymorphism in Takayasu's arteritis.

OBJECTIVE: Takayasu's arteritis (TA) is a chronic, rare granulomatous panarteritis of unknown aetiology involving mainly the aorta and its major branches. In this study, genetic susceptibility to TA has been investigated by screening the functional single nucleotide polymorphism (SNP) of PTPN22 gene encoding the lymphoid-specific protein tyrosine phosphatase. METHODS: Totally, 181 patients with TA and 177 healthy controls are genotyped by PCR-RFLP method for the SNP rs2476601 (A/G) of PTPN22 gene. Polymorphic region was amplified by PCR and digested with Xcm I enzyme. RESULTS: Detected frequencies of heterozygous genotype (AG) were 5.1% (9/177) in control group and 3.8% (7/181) in TA group (P = 0.61, odds ratio: 0.75, 95% CI: 0.3, 2.0). No association with angiographic type, vascular involvement or prognosis of TA was observed either. CONCLUSION: The distribution of PTPN22 polymorphism did not reveal any association with TA in Turkey.

The role of HLA-DRB1 shared epitope alleles in predicting short-term response to leflunomide in rheumatoid arthritis.

OBJECTIVES: To investigate the role of shared epitope (SE) alleles in the short-term clinical response to leflunomide for the treatment of active RA. METHODS: In an open-label, multi-centre study of 16-weeks duration, 93 patients (82% female) fulfilling ARA 1987 RA criteria were treated with leflunomide (100 mg loading dose for 3 days, then 20 mg/day as the maintenance dose). The primary efficacy criterion was the response status according to the European League Against Rheumatism (EULAR) response criteria using Disease Activity Score-28 (DAS28) activity measure. SE determinations have been undertaken by polymerase chain reaction and sequence-specific oligonucleotide genotyping methods. RESULTS: The mean (s.d.) Disease Activity Score-28 (DAS28) was 5.1 (1.3) before the treatment, which was significantly decreased after 16 weeks [3.0 (1.1), P < 0.001]. According to the EULAR response criteria, 55 patients (59.1%) were classified as good responders. SE was positive in 51 (54.8%) of the patients, with 13 (13.9%) having SE homozygosity or carrying any two SE alleles. Among SE-positive patients, 68.6% (35/51) were good responders, compared with 47.6% (20/42) in SE negatives (P = 0.04). No difference was present according to SE hetero- or homozygosity (68.4 vs 69.2%). RF was also present significantly more frequently in the SE-positive group compared with negatives (78.4 vs 57.1%, P = 0.03). However, no significant difference was observed in the prevalence of RF positivity in patients with a good clinical response (72.7 vs 63.2%, P = 0.32). CONCLUSIONS: The results suggest that HLA-DRB1 SE presence may favourably affect the outcome of leflunomide monotherapy in an unselected group of RA patients with an active disease and naive to leflunomide.

PTPN22 gene polymorphism in Behçet's disease.

A functional single nucleotide polymorphism (SNP) of PTPN22 gene encoding the protein tyrosine phosphatase has been reported to be associated with autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus and type I diabetes. PTPN22 R620W polymorphism has a wide variation of allelic frequencies among different populations. This polymorphism is investigated in Turkish patients with Behçet's disease (BD), a systemic vasculitis with immune activation. DNA samples from 134 patients with BD and 177 healthy controls are genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism method for the SNP (rs2476601, A/G) of PTPN22 gene. Polymorphic region was amplified by PCR and digested with XcmI enzyme. The frequency of heterozygous genotype (AG) was 5.1% (9/177) in control group, whereas polymorphic allele was not present in the whole BD group (P = 0.012, OR 0.65, 95% confidence interval 0.0-1.1). Both the lower prevalence in the general population and the absence in BD show the limited role of PTPN22 polymorphism in the pathogenesis of autoimmunity in Turkey.

Relative predispositional effects of HLA class II DRB1-DQB1 haplotypes and genotypes on type 1 diabetes: a meta-analysis.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

Clinical comparison of anti-MuSK- vs anti-AChR-positive and seronegative myasthenia gravis.

We compared 65 anti-acetylcholine receptor (AChR)-negative myasthenia gravis (MG) patients, including 32 anti-muscle-specific tyrosine kinase (MuSK)-positive (49%) and 33 anti-MuSK-negative (seronegative) (51%) patients, with 161 anti-AChR-positive MG patients. The anti-MuSK-positive group had a higher frequency of bulbar involvement and respiratory crises. The seronegative group was in between the anti-MuSK positive and the anti-AChR positive groups, being closer to the latter, with regard to the severity of the disease. At the end of follow-up, the outcome of the anti-MuSK-positive patients was not different from that of the anti-AChR-positive patients, although their maintenance corticosteroid dose was higher. The seronegative patients had better outcome than the other two groups.

Interleukin (IL)-12, IL-2, and IL-6 gene polymorphisms in Takayasu's arteritis from Turkey.

Takayasu's arteritis (TA) is a chronic arterial inflammation of unknown etiology involving mainly the aorta and its major branches. Genetic polymorphisms of cytokines are screened as susceptibility factors for TA in Turkey. A total of 94 patients with TA were investigated for the genetic polymorphisms of the interleukin genes IL12, IL2,and IL6 and were compared with 108 healthy control subjects using polymerase chain reaction-sequence-specific primer method. The frequencies of IL12B 1188 C allele (p = 0.03, OR = 1.7) and CC genotype (p = 0.007, OR = 3.7) were both higher in TA patients than in control subjects. TT genotype at IL2-330 (p = 0.006, OR = 2.4) and GG genotype at IL6-174 (p = 0.04, OR = 1.9) were more frequent in TA patients. Lower prevalence of GT genotype at IL2-330 (p = 0.005, OR = 0.4), CG genotype at IL6-174 (p = 0.001, OR = 0.4), and AG genotypes at IL6-598 (p = 0.01, OR = 0.4) were also detected. The polymorphism of IL-12 as well as IL-6 and IL-2 genes may contribute to susceptibility and pathogenesis of TA by altering cytokine production and inducing inflammation.