Detection of Shigella spp. nucleic acids in the synovial tissue of Tunisian rheumatoid arthritis patients and other forms of arthritis by quantitative real-time polymerase chain reaction.

Enterobacterial components in the joints of patients are believed to contribute to a perpetuating inflammation leading to a reactive arthritis (ReA), a condition in which microbial agents cannot be recovered from the joint. At present, it is unclear whether nucleic acids from Shigella spp. are playing a pathogenic role in causing not only ReA but also other forms of arthritis. Quantitative real-time polymerase chain reaction assay (qPCR) is the method of choice for the identification of bacteria within the synovium. The aim of our study was to detect the presence of Shigella spp. nucleic acids in the synovial tissue (ST) of Tunisian arthritis patients. We investigated 57 ST samples from rheumatoid arthritis (RA) n = 38, undifferentiated oligoarthritis (UOA) n = 12, and spondyloarthritis (SpA) n = 7 patients; 5 ST samples from healthy individuals were used as controls. Shigella spp. DNA and mRNA transcripts encoding the virulence gene A (VirA) were examined using an optimized qPCR with newly designed primers and probes. Using qPCR, Shigella spp. DNA was found in 37/57 (65%) ST samples (24/38, i.e., 63.2% of RA, 8/12, i.e., 67% of UOA, and 5/7, i.e., 71.4% of SpA patients). Paired DNA and mRNA were extracted from 39 ST samples, whose VirA cDNA was found in 29/39 (74.4%) patients. qPCR did not yield any nucleic acids in the five healthy control ST samples. The qPCR assay was sensitive and showed a good intra- and inter-run reproducibility. These preliminary findings generated by an optimized, highly sensitive PCR assay underline a potential role of past gastrointestinal infections. In Tunisian patients, a bacterial etiology involving Shigella spp. in the manifestation of arthritic disorders including RA might be more common than expected.

Unilateral hypertrophic osteoarthropathy in a patient with a vascular graft infection.

We report a patient who presented with high fever; a unilateral, palpable tender swelling along the right shin; and effusions in knee and ankle joints leading to the diagnosis of hypertrophic osteoarthropathy (HOA). The diagnostic workup revealed an infected vascular graft that had been implanted 2 years before because of a ruptured infrarenal aortic aneurysm as the cause of HOA. The patient was treated successfully with antibiotics and surgically replacing the infected graft. Hypertrophic osteoarthritis is a clinical entity characterized by digital clubbing, periostitis, and synovial effusions. Primary and secondary forms have been described. Secondary HOA develops as a consequence of various diseases, mainly intrathoracic malignancies. Vascular graft infection, as reported here, is a rare cause of HOA. This case underlines the typical clinical features of HOA and the importance of a prompt and comprehensive diagnostic workup in cases of HOA. Our aim is to sharpen the awareness of its multiple underlying causes. Unilateral HOA is a rare but strong and important sign of infection of vascular prosthesis.

Association and frequency of HLA-A, B and HLA-DR genes in south Tunisian patients with spondyloarthritis (SpA).

The aim of this study is to investigate the association of HLA-A, B and HLA-DR gene expression and to assess an association of additional HLA antigens besides HLA-B27 in south Tunisian patients with spondyloarthritis (SpA). Eighty-five patients diagnosed with ankylosing spondylitis (AS, n=68) and reactive arthrithis (ReA, n=17) were selected and compared with 100 healthy controls (HC). HLA class I antigens were typed serologically using microlymphocytotoxicity technique. HLA-DRB1* alleles were studied by polymerase chain reaction amplification with sequence-specific primers. The significance of differences between patients and controls was tested by chi-square analysis. We found significantly increased frequencies of HLA-A3 (30.6%; pC=0.04; OR=2.95), HLA-B27 (62.35%; pC=4.10(-17), OR=53.55), and HLA-DRB1*15 (17.2%; pC=0.026; RR=2.58) alleles in SpA patients compared to HC. The most frequent and strongest association was observed for HLA-B27 in AS (pC=6.6 ×10(-16), OR=52.23). When AS and ReA patients were analysed separately, HLA-DRB1*15 and HLA-A3 were increased only in AS (pC=0.01, OR=2.99 and pC=0.03, OR=3.14, respectively). In ReA patients, HLA-DRB1*04 (p=0.033, pC=NS, OR=2.89) was found to be the most common allele. By analysing the HLA-B27-negative subgroup, HLA-A3 and HLA-DRB1*15 expression was found to be dependent on the presence of HLA-B27. HLA-B27 expression was higher in male (45/53; 85%) as compared to female (8/53; 15%) patients (p=0.03). Apart from HLA-B27, HLA-A3 and HLA-DRB1*15 are the MHC class I and II alleles found most frequent in Tunisian patients with AS, whereas HLA-DRB1*04 was found most frequent in ReA patients. HLA-B27 is more frequent in male than in female patients.

Cost of illness in rheumatoid arthritis in Germany in 1997-98 and 2002: cost drivers and cost savings.

OBJECTIVE: Comparison of overall RA-related costs and of relative contribution of single-cost domains before and after the introduction of TNF-blocking agents in Germany. METHODS: Two cohorts of RA outpatients (ACR '87 criteria) with long-standing disease are assessed in terms of disease-related costs and cost composition (n = 106 patients in 1997-98 and n = 180 patients in 2002 with similar patient characteristics). Full-cost analyses are performed including direct disease-related costs (medical and non-medical) and productivity costs as collected by patient questionnaires. Absolute costs (€/patient/year) are compared and the impact of single-cost domains on overall costing in RA is estimated (relative proportions of cost components within samples). RESULTS: Overall costs are comparable (1997-98: €4280; 2002: €3830; not significant). Differences can be observed in medication (1997-98: €550; 2002: €1580; P < 0.001) and hospitalization costs (1997-98: €1240; 2002: €500; P < 0.001). Productivity costs are significantly lower (€1480 vs €850; P < 0.05) in 2002. The impact of medication costs is outstanding in the 2002 sample (42 vs 12%), the proportion of hospitalization costs is substantially lower (29 vs 13%). Costs for DMARDs in 2002 are mostly driven by TNF blockers (37%). The number of DMARDs per patient is higher in 2002 as are costs for osteoporosis medication and gastroprotective treatment. CONCLUSION: Although overall costs before and after the introduction of TNF blockers are comparable, the decrease in hospitalization and productivity costs is promising in terms of future long-term cost savings. The development of these aspects and of the increasing medication costs will have to be evaluated with longer time frames.

Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis.

INTRODUCTION: Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. METHODS: We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. RESULTS: Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. CONCLUSIONS: Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.

Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis.

INTRODUCTION: We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment. Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF). METHODS: mRNA expression levels of four ligands (NGF, brain derived growth factor (BDNF), neurotrophin (NT)-3, NT-4) and their four corresponding receptors (tyrosine kinase (trk) A, trkB, trkC, NGFRp75) were determined in the synovial fluid (SF) cells of 9 patients with rheumatoid arthritis (RA) and 16 with spondyloarthritis (SpA) and compared with 7 osteoarthritis (OA) patients. NGF was also determined in synovial tissue (ST) biopsies of 10 RA and 10 SpA patients. The production of NGF by monocytes and lymphocytes was assessed by flow cytometry of SF cells, synovial tissue derived fibroblast-like synoviocytes (FLS) were assessed by ELISA on culture supernatant. RESULTS: SF cell analysis revealed a clear BDNF and NGF mRNA expression, with significantly higher NGF expression in RA and SpA patients than in the OA group. NGF expression was higher in ST samples of RA as compared to SpA. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ T lymphocytes and CD14+ cells, i.e. monocytes/macrophages, whereas cultured FLS did not produce NGF in vitro. CONCLUSIONS: Neurotrophins and especially NGF are expressed in the synovial fluid and tissue of patients with peripheral synovitis. The presence of neurotrophins as well as their receptors, in particular the NGF/trkA-p75 axis in peripheral synovitis warrants further functional investigation of their active involvement in chronic inflammatory arthritis.

Identification of candidate genes for susceptibility to reactive arthritis.

This study was undertaken to evaluate the gene expression profile in monocytes from three patients with reactive arthritis (ReA) in remission in order to identify candidate genes accounting for a potential susceptibility to ReA. Gene expression analyses revealed eight differentially expressed mRNA transcripts in monocytes of ReA patients. The major part of genes encoded cytokines, growth factors and chemokines. There was a remarkably high proportion of proangiogenic factors, in particular IP10, ENA-78, and IL-8 accounting for a genetically determined susceptibility to ReA at the host cell level.

Treatment of sicca symptoms with hydroxychloroquine in patients with Sjogren's syndrome.

OBJECTIVE: There is no established disease-modifying treatment of xerostomia and xerophthalmia in SS. This retrospective study was performed in order to evaluate the efficacy of HCQ for glandular function, i.e. saliva and tear production. METHODS: Fourteen patients with primary SS (pSS) were included (Group A). All patients were anti-Ro and/or -La antibody positive except one. Patients were treated with HCQ for a period of up to 6 months. Glandular function was determined by Saxon's and Schirmer's tests for the dominant eye at baseline and at the end of the treatment. We included a control group of 21 patients with objective sicca symptoms and positive alpha-fodrin antibodies (Group B). RESULTS: In patients with pSS (Group A), a significant increase in saliva production after HCQ treatment (P = 0.022) was observed. A subanalysis revealed that particularly the alpha-fodrin-positive patients responded to HCQ (P = 0.017 alpha-fodrin positive vs P = 0.4 alpha-fodrin negative). Interestingly, patients with sicca symptoms and alpha-fodrin antibodies (Group B) showed a significant increase in tear production (P = 0.001). In addition, there was a positive correlation between the alpha-fodrin IgA antibody concentration and the Schirmer's test at baseline (r = 0.66; P = 0.001) and after treatment (r = 0.6; P = 0.004) in this group. CONCLUSIONS: HCQ treatment led to a beneficial effect on xerostomia in patients with pSS who lack severe organ manifestations. The response was greater in alpha-fodrin-positive patients.

Detection and frequency of Chlamydia trachomatis DNA in synovial samples from Tunisian patients with reactive arthritis and undifferentiated oligoarthritis.

We aimed to determine the frequency of Chlamydia trachomatis DNA in the synovial compartment of 34 arthritic patients. Chlamydia trachomatis DNA was detected using a nested PCR targeting the cryptic plasmid, the 16S rRNA gene and the outer membrane protein 1 gene. The presence of serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis was studied by a microimmunofluorescence assay and by an enzyme-linked immunosorbent assay, respectively. Synovial samples from 20 of 34 (59%) patients [nine with reactive arthritis (ReA), seven with undifferentiated oligoarthritis (UOA), two with rheumatoid arthritis and two with osteoarthritis] were positive for at least one C. trachomatis DNA sequence by nested PCR. The high sensitivity results most likely from the combination of a standardized automated MagNA Pure extraction method, PCR targeting three different C. trachomatis genes and the screening for C. trachomatis in synovial tissue and fluid samples. There was no correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia-specific serologic response. Our data support that PCR is the method of choice to establish the diagnosis of Chlamydia-induced arthritis in patients with ReA. We suggest that this diagnosis might also be considered in C. trachomatis-positive patients previously classified as UOA.

Identification of interleukin-7 as a candidate disease mediator in spondylarthritis.

OBJECTIVE: Understanding of the molecular pathophysiology of spondylarthritis (SpA) remains largely elusive. This is related both to the complexity of the disease (axial versus peripheral disease, inflammation versus tissue remodeling) and to the difficulty in obtaining samples from primary disease sites. This study was undertaken to explore a gene expression approach for identifying novel candidate mediators of SpA. METHODS: Sacroiliac joint fluid aspirates from 3 SpA patients with active sacroiliitis were studied by microarray analysis. The expression of selected candidate molecules in peripheral synovitis was confirmed by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Microarray analysis identified 4 sacroiliitis gene clusters, containing a total of 47 messenger RNA (mRNA) transcripts. Two clusters contained genes expressed in all sacroiliitis samples, corresponding to both known and unsuspected candidate mediators of SpA pathology. These included proinflammatory molecules as well as molecules involved in tissue remodeling, such as transforming growth factor beta2. Of the novel candidate genes selected for confirmation, interleukin-7 (IL-7) mRNA expression was higher in SpA peripheral synovial fluid and synovial tissue samples than in osteoarthritis samples, and similar to expression in rheumatoid arthritis (RA) samples. At the protein level, synovial fluid IL-7 levels were even higher in SpA than in RA, despite lower levels of tumor necrosis factor alpha and IL-1beta. CONCLUSION: In the present study, both known and unsuspected candidate mediators of SpA pathogenesis were identified, including IL-7. The specific overexpression of IL-7 at sites of peripheral synovitis in SpA suggests that further functional investigations of the role of this cytokine in SpA pathogenesis are warranted.

Acute coronary syndrome associated with Churg-Strauss syndrome.

A 41 -year old female patient was admitted with acute onset of dyspnea and chest pain. Previous history revealed asthma, chronic sinusitis and eosinophilic proctitis. Electrocardiogram showed anterior ST-segment elevations and inferior ST-segment depression. Immediate heart catheterization revealed a distally occluded left anterior descending coronary artery, the occlusion being reversible after nitroglycerine. Cardiac magnetic resonance imaging was consistent with perimyocarditis. Hypereosinophilia and IgE elevation were present and Churg-strauss syndrome was diagnosed.

Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

The aim of this study was to perform a comprehensive gene expression analysis of cytokines, chemokines, and their receptors in Chlamydia trachomatis-infected human monocytes in order to elucidate molecular aspects of their involvement in the host response. Peripheral blood mononuclear cells from three healthy donors were separated and infected with C. trachomatis elementary bodies serovar K (UW/31/Cx) at a multiplicity of infection of 5:1. Three time points of infection were studied by gene expression analysis using microarray: 4 hours (active infection), 1 day (transition), and 7 days (persistent infection). Expression levels of selected genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Transcripts encoding 10 cytokines, chemokines, and receptors were found to be upregulated exclusively in the early, active phase of the infection as compared to four genes in the late, persistent state of the infection. Apart from receptors, both the level and the number of transcripts encoding inflammatory products decreased with ongoing infection. Four genes (interferon-gamma, macrophage inflammatory protein [MIP]-1-alpha, MIP-1-beta, and interleukin-2 receptor-gamma) were constantly expressed over a period of 7 days. The current study provides data on the induction of mRNA encoding cytokines, chemokines, and their receptors in C. trachomatis-infected human monocytes. This pro-inflammatory gene expression profile of the monocytic host cell showed several differences between active and persistent chlamydial infections.

Infection and musculoskeletal conditions: Reactive arthritis.

Reactive arthritis (ReA) has been recognized as a clinical disease entity for nearly 100 years. The prevalence is estimated to be 30-40/100,000 adults. The HLA-B27-associated form is part of the spondyloarthritis concept. According to the current hypothesis the arthritis follows a primary extra-articular infection and is characterized by the presence of bacterial antigen and/or of viable but non-culturable bacteria persisting within the joint. Pathogenesis involves the modification of host cells by pathogen-associated molecular patterns (PAMPs, e.g. lipopolysaccharide), bacterial effector proteins, the adaptive immune system, and the genetic background. Up to 30% of patients develop chronic symptoms, and therapeutic options for these patients are still limited. Data for recommendations to apply conventional disease-modifying anti-rheumatic drugs (DMARDs) are rare; however, sulfasalazine seems to be effective, and first reports on agents that block tumour necrosis factor (TNF) are promising. Combination therapy of several antibiotics might open the window to curing the disease; however, controlled clinical studies are needed.

Successful treatment of post-kala-azar dermal leishmaniasis (PKDL) in a HIV infected patient with multiple relapsing leishmaniasis from Western Europe.

We present a 42-year-old man who was admitted with worsening of his general condition and facial skin lesions. He had previously been diagnosed with HIV infection and visceral leishmaniasis (VL). Diagnostic work-up revealed a new relapse of VL paralleled by the diagnosis of post-kala-azar dermal leishmaniasis (PKDL). The patient was treated with IV liposomal amphotericin B as well as sodium stibogluconate followed by oral hexadecylphosphocholine (miltefosine) over a period of 9 months. PKDL lesions began to disappear after 8 months of treatment. In addition, severe and relapsing VL so far remains in remission. This case demonstrates successful treatment of PKDL and relapsing VL in a Western European patient with HIV infection.

Serum levels of matrix metalloproteinase 3 and macrophage colony-stimulating factor 1 correlate with disease activity in ankylosing spondylitis.

OBJECTIVE: To assess the usefulness of measuring serum matrix metalloproteinase 3 (MMP-3) and macrophage colony-stimulating factor (M-CSF) in patients with ankylosing spondylitis (AS). METHODS: Serum levels of MMP-3 and M-CSF were measured in AS patients who did and did not receive infliximab treatment. These were compared with those of 28 healthy subjects. RESULTS: In the group of AS patients not treated with biologics, both M-CSF and MMP-3 correlated with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) values, but not with each other. Logistic regression analysis showed that MMP-3 values were high in those with severely active disease. Infusions of infliximab in AS patients led to a significant decrease in the values of the BASDAI as well as the serum MMP-3, but no change in the serum M-CSF values. CONCLUSION: MMP-3 and M-CSF are potentially useful markers of AS disease activity.

Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down-regulation by tumor necrosis factor alpha blockade in spondylarthropathy.

OBJECTIVE: To investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in spondylarthropathy (SpA) synovitis. METHODS: Paired samples of synovial biopsy tissue as well as serum and synovial fluid (SF) from 41 patients with SpA and 20 patients with rheumatoid arthritis (RA) and serum samples from 20 healthy controls were analyzed by immunohistochemistry and enzyme-linked immunosorbent assay for the presence of MMPs 1, 2, 3, and 9 and TIMPs 1 and 2. In addition, sera from 16 patients with ankylosing spondylitis (AS) and peripheral synovitis and 17 patients with AS and exclusively axial involvement were analyzed. An additional cohort of SpA patients was analyzed at baseline and after 12 weeks of infliximab treatment. RESULTS: Staining for MMPs and TIMPs showed a cellular and interstitial pattern in the synovial lining and sublining layers that was similar between the RA and SpA patients. Involvement of MMPs and TIMPs in SpA synovitis was suggested by the correlation with cellular infiltration, vascularization, and cartilage degradation. Higher serum levels of MMPs 3 and 9 were revealed in SpA and RA patients as compared with healthy controls. Production of MMP-3, but not MMP-9, in the serum reflected the presence of peripheral synovitis, as indicated by 1) the correlation between serum levels, SF levels (which were 1,000-fold higher than the serum levels), and synovial expression of MMP-3, 2) the increased levels of MMP-3 in AS patients with peripheral disease and not exclusively axial involvement, and 3) the correlation of serum and SF MMP-3 with parameters of synovial, but not systemic, inflammation. The modulation of the MMP/TIMP system by tumor necrosis factor alpha (TNFalpha) blockade was confirmed by the down-regulation of all MMPs and TIMPs in the synovium and a pronounced and rapid decrease of serum MMP-3. CONCLUSION: MMPs and TIMPs are highly expressed in SpA synovitis and mirror both the inflammatory and tissue-remodeling aspects of the local disease process. Serum MMP-3, originating from the inflamed joint, represents a valuable biomarker for peripheral synovitis. Modulation of the MMP/TIMP system by infliximab could contribute to the antiinflammatory and tissue-remodeling effects of TNFalpha blockade in SpA.