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While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4+ T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4+ T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their in vivo antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols., Graphical Abstract, Despite HBV vaccination failure in end-stage renal disease (ESRD), HBV-reactive T cells are generated in about 70% of ESRD-vaccinees. Further, the non-responder group demonstrated polyfunctional HBV-reactive CD4+ T cells suggesting their potential anti-viral capacities. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols.

Analysis of interleukin (IL)-6 serum levels in patients with ankylosing spondylitis (AS) has indicated that IL-6 might be a pro-inflammatory cytokine involved in AS. However, two placebo-controlled trials with monoclonal antibodies directed against the IL-6 receptor have failed to demonstrate the efficacy of the monoclonal humanized anti-human IL-6 receptor antibody over placebo for the treatment of symptoms of AS. In this study we conducted an in situ analysis of IL-6 expression at different sites of inflammation in zygapophyseal joints of patients with AS in comparison to osteoarthritis autopsy controls (CO).Our immunohistochemical analysis involved 14 patients with AS, 12 autopsy controls (CO), and 11 patients with osteoarthritis (OA). Immunohistochemistry was performed to detect IL-6+ cells at five different sites: within subchondral bone marrow, fibrous tissue replacing subchondral bone marrow, hyaline cartilage, and the subchondral bone plate, and at entheseal sites.Apart from changes in subchondral bone marrow, no significant differences were observed at the sites analysed when comparing AS patients and controls. A significantly lower frequency of IL-6+ cells was evident in AS patients compared to controls (p = 0.0043). In addition, AS patients tended to have even lower percentages of IL-6+ cells than controls at subchondral bone plates and entheseal sites. A significantly lower number of IL-6 expressing cells was also seen within the fibrous tissue of AS compared to OA patients (p = 0.0237).This in situ analysis confirms that IL-6 is not a key player in the pathogenesis of inflammatory processes in spondyloarthritides (SpA). The relevance of pro-inflammatory agents in axial SpA might be studied better in situ in bony specimens at the primary site of inflammation.

Objective To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS). Methods Histomorphologic and histomorphometric analyses were performed on zygapophyseal joints obtained from 18 patients with AS, 9 patients with osteoarthritis (OA), and 10 cadaver donors without a rheumatic disease (controls). The proteoglycan content of the cartilage was determined by Safranin O staining and the chondrocyte apoptosis according to caspase 3 expression. Results AS joints were categorized into 3 groups according to the morphology of the joint surfaces and joint space: group 1 were joints with an open joint space, group 2 were joints with cartilaginous fusion, and group 3 were joints with bony fusion of the joint surfaces. Progressive loss of the joint space from group 1 joints to group 3 joints suggests that this grouping corresponds to sequential stages of joint remodeling. Cartilage thickness and subchondral bone plate thickness declined from group 1 to group 3 (P < 0.01). Increased chondrocyte apoptosis rates were found in groups 1 and 2 (P < 0.05), while in group 3, a reduction in the proteoglycan content was found (P < 0.001). Bone marrow replacement and invasion of the subchondral bone plate by fibrous tissue was found predominantly in AS joints in group 2. Conclusion Cartilage degeneration, indicated by cartilage thinning, enhanced chondrocyte apoptosis, and proteoglycan loss, and subchondral bone thinning, promoted by invasion of the subchondral bone plate by a fibrous tissue originating from the bone marrow, are hallmarks of joint remodeling in AS.

Ankylosing spondylitis (AS) is a chronic inflammatory disease predominantly affecting the axial skeleton, leading potentially to bone erosions, new bone formation, and ankylosis of the spine. The burden of disease is determined by the grade of acute inflammation causing pain and stiffness and by new bone formation causing a reduction in spinal mobility1. Histopathological studies from intervertebral discs2, femoral heads3,4, sacroiliac joints5,6, manubriosternal junction7, and zygapophyseal joints8,9,10 enabled us in the past to define characteristic histomorphological and immunohistochemical features of acute inflammation in bony samples from patients with AS: enthesitis and subchondral inflammation at the interface between bone and cartilage — subchondral osteitis — are the primary events in AS immunopathology. Cytokines such as tumor necrosis factor-α (TNF-α)5, interleukin 17 (IL-17)11, and IL-239 seem to be triggering factors during inflammatory processes. The rather unexpected observation that TNF-α blocking therapy did not reduce new bone formation and radiographic progression in patients with AS underscored the need for a better pathophysiological understanding of osteoproliferative mechanisms in AS. Currently, investigations are focused on 2 major hypotheses. The first is that new bone formation is an independent feature uncoupled from inflammation triggered by mechanical stress. Microdamage and/or cell stress at the enthesial site trigger both an inflammatory and an anabolic process in the bone, i.e., through bone morphogenic protein signaling, leading to typical clinical signs of AS12,13. The second is that new bone formation is closely linked to inflammation through initial … Address correspondence to Dr. H. Appel, Charite Universitatsmedizin Berlin, Campus Benjamin Franklin, Rheumatology, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: heiner.appel{at}charite.de

Objective To investigate the role of serum vascular endothelial growth factor (VEGF) as a predictor of radiographic spinal progression in patients with axial spondyloarthritis (axSpA). Methods Altogether, 172 patients with definite axSpA (95 with ankylosing spondylitis and 77 with non-radiographic axSpA) were included in this study. Spinal radiographs obtained at baseline and after 2 years of follow-up were scored independently by two trained readers in a concealed and randomly selected order according to the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) scoring system and for the presence of syndesmophytes. Radiographic spinal progression after 2 years was defined as (1) mSASSS worsening by ≥2 units, and (2) new syndesmophyte formation or formation of a bridging syndesmophyte from two single syndesmophytes. Serum VEGF levels were detected at baseline. Results Mean baseline VEGF values were significantly higher in patients with mSASSS worsening by ≥2 units after 2 years (n=22) than in those without progression (562±357 vs 402±309 pg/mL, respectively, p=0.027) and in patients with syndesmophyte formation (n=18) again as compared with those without new bone formation (579±386 vs 404±307 pg/mL, respectively, p=0.041). VEGF as a predictor of radiographic spinal progression performed especially well in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n=48). In these patients, a VEGF serum level of >600 pg/mL had a sensitivity of 53%, a specificity of 97% and an OR=36.6 (95% CI 3.9 to 341.5) as a predictor of mSASSS worsening by ≥2 units. For syndesmophyte formation, elevated VEGF demonstrated a sensitivity of 47%, a specificity of 94% and an OR=13.6 (95% CI 2.4 to 78.3). Conclusions An elevated serum level of VEGF (>600 pg/mL) is highly specific as a predictor of radiographic spinal progression in patients with axSpA, especially in patients who are at high risk for further progression due to the presence of syndesmophytes.

Objective The interleukin-12 (IL-12) family of cytokines has been suggested to play a critical role in inflammatory autoimmune diseases, and recent studies analyzing peripheral blood and synovial fluid from patients with spondyloarthritides suggest that IL-23 might be a proinflammatory factor in these disorders. This study was undertaken to investigate the presence and source of IL-23 in the spines of patients with ankylosing spondylitis (AS). Methods The frequency of IL-23–positive and IL-12–positive cells within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of patients with AS was analyzed by immunohistochemistry. The origin of IL-23–positive cells was determined by double staining of CD163+ macrophages, CD68+ macrophages, CD1a+ dendritic cells, tryptase-positive mast cells, myeloperoxidase-positive cells, CD20+ B cells, and CD3+ T cells. Findings in 28 facet joints from 22 AS patients were compared with those in 20 facet joints from 13 patients with osteoarthritis (OA) and 10 normal control specimens. Results The frequency of IL-23–positive cells in subchondral bone marrow from the joints of AS patients (mean ± SD 42.50 ± 32.81/high-power field [hpf]) was significantly increased compared to that in subchondral bone marrow from OA patients (OA 15.63 ± 29.90/hpf) (P = 0.0017) or controls (19.36 ± 16.8/hpf) (P = 0.03). Myeloperoxidase-positive cells and, to a lesser extent, macrophages and dendritic cells were found to be the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23–positive cells was significantly higher than that of IL-12–positive cells (P < 0.001 in both patient groups). Within fibrous tissue from AS and OA facet joints, IL-23 was predominantly produced by CD163+ macrophages (mean ± SD 0.64 ± 0.59/hpf and 4.36 ± 3.4/hpf, respectively) and CD68+ macrophages (2.3 ± 0.65/hpf and 6.54 ± 4.1/hpf, respectively). Conclusion IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of patients with AS. It might have a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting potential therapeutic target in this disease.

Objective NSAIDs are first-line therapy in axial SpA (axSpA). The proportion of patients responding to NSAIDs and differences between AS and non-radiographic axSpA (nr-axSpA) in this regard have not been studied in detail to date. The aim of this study was to examine the proportion of patients with AS and nr-axSpA responding to NSAIDs according to current treatment recommendations. Methods Consecutive anti-TNF-naive patients with nr-axSpA and AS (n = 50 each) were included if their BASDAI score was ⩾4 without having received maximal NSAID doses. In case of a BASDAI score ⩾4 1 week later, another NSAID was prescribed. For the next 3 weeks, continuous intake of maximal doses was recommended but patients could reduce doses in case of intolerance or improvement. MRI of the SI joints was performed at baseline and week 4. Results All outcomes except for CRP and MRI scores improved significantly after 4 weeks of NSAIDs, with no difference between axSpA subgroups. An Assessment of SpondyloArthritis international Society 40% (ASAS40) response and partial remission rates were 35 and 16% at week 4, respectively. At the same time point, a BASDAI score ⩾4 was still present in 44% of patients, 30% of which had reduced NSAID doses, partly due to intolerance (38%). Only 13% of all patients had continuously taken NSAIDs at the maximal dosage, but there was no difference in the efficacy outcome compared with those who had taken reduced doses. Conclusion AS and nr-axSpA patients had similar response rates to NSAIDs while objective signs of inflammation did not change over 4 weeks. Only a minority of patients was willing to take maximal doses of NSAIDs, and ⩾40% patients remained candidates for TNF blockers. These results may influence future trial designs.

Seit Einfuhrung der Biologika sind Biomarker zur Fruhdiagnostik und Therapieentscheidung von zunehmender Bedeutung. Sie sollen im BMBF-geforderten Forschungsverbund ArthroMark fur die rheumatoide Arthritis und die Spondylarthritis etabliert werden. Biobanken und Vorarbeiten zu Genotypen, Genexpressions- und Autoantigenprofilen dienen als Grundlage. Bioinformatische Vernetzung soll die Untersuchungen aufeinander abstimmen und eine Studie mit modernen Bildgebungsverfahren die funktionelle Bedeutung der neuen Biomarker bestmoglich charakterisieren. Zur Validierung der diagnostischen Marker wird eine schrittweise Erweiterung des Netzwerkes angestrebt.

Recent studies using magnetic resonance imaging have suggested that the subchondral bone marrow and the entheses are the sites which are primarily involved in the peripheral and axial inflammation found in patients with spondyloarthritides. Histopathological analyses indicated that the typical morphological features at these sites reflect an inflammation (osteitis) at the bone cartilage interface and in the subchondral bone marrow. This finding implies that synovitis may be of minor importance, especially in comparison to other inflammatory joint diseases such as rheumatoid arthritis. Here, we summarize current available knowledge on synovial involvement in inflammatory processes related to SpA.

Objective Histologic studies have shown B cell clusters in the subchondral bone marrow of the spine of patients with ankylosing spondylitis (AS). An immunotherapy targeting B cells in AS is therefore of interest. We undertook this study to examine the efficacy and safety of rituximab in patients with AS refractory to nonsteroidal antiinflammatory drugs in whom previous treatment with tumor necrosis factor α (TNFα) blockers either had not been tried or had failed. Methods In this phase II clinical trial, 1,000 mg rituximab was administered intravenously at baseline and at week 2 in 20 patients with active AS. Ten of these patients had never received TNF blockers, and treatment with TNF blockers had failed in the other 10 patients. The primary end point was a 20% improvement in disease activity at week 24 according to the criteria of the Assessment of SpondyloArthritis international Society (an ASAS20 response). Results Seventy-five percent of the patients were male, 90% were HLA–B27 positive, their mean age was 39.7 years, and their mean disease duration was 16.8 years. Patients had active disease, defined as a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score ≥4. While there was no clear response at week 24 in the group in whom TNF blockers had failed (30% had achieved an ASAS20 response, 10% had achieved an ASAS40 response, none had achieved partial remission according to the ASAS criteria, and none had achieved 50% improvement on the BASDAI [a BASDAI50 response] beyond an expected placebo response), we observed a good improvement in the TNF blocker–naive group at week 24 (50% had achieved an ASAS20 response, 40% had achieved an ASAS40 response, 30% had achieved partial remission according to the ASAS criteria, and 50% had achieved a BASDAI50 response). Conclusion Although rituximab does not seem to be effective in patients with AS that does not respond to TNF blockers, it had significant efficacy in TNF blocker–naive patients. Therefore, further controlled trials with B cell–directed therapies should be performed in TNF blocker–naive AS patients in the future.

Objective.New bone formation of the spine is a typical feature of ankylosing spondylitis (AS). It is unknown whether new bone formation is part of a physiological repair process or a unique pathological entity of the disease.Methods.We analyzed zygapophyseal joints from patients with AS and osteoarthritis (OA) undergoing spinal surgery for rigid hyperkyphosis (AS) or radiculopathy caused by severe OA. In 17 patients with AS, 11 with OA, and 5 controls we performed immunohistochemical analysis of osteoprotegerin (OPG), nuclear factor-κB ligand (RANKL), and osteocalcin (OC) expression in osteoblasts and determined the trabecular thickness in AS and OA patients and controls. Osteoclasts were detected by tartrate-resistant alkaline phosphatase (TRAP) staining.Results.Trabecular thickness was significantly lower in patients with AS compared to OA (p = 0.01). The absolute number of CD56+ osteoblasts (p < 0.001) and OC+ (p = 0.002), OPG+ (p = 0.003), and RANKL+ osteoblasts (p = 0.03) in AS patients was also significantly lower than in OA patients. The percentages of OC+, OPG+, and RANKL+ osteoblasts did not differ between AS and OA (p > 0.05 in all cases). In controls, the percentages of OPG+ (p = 0.013) and OC+ (p = 0.034) but not RANKL+ (p > 0.05) osteoblasts were significantly lower compared to AS patients. The frequency of TRAP+ osteoclasts in AS patients was significantly lower compared to OA (p < 0.001), but higher compared to controls.Conclusion.Immunohistochemical analysis of zygapophyseal joints suggested that osteoblast activity is similar in AS and OA, indicating that new bone formation is possibly a physiological function of repair in both diseases.

Background Imaging has an essential role in the new spondyloarthritis (SpA) classification criteria for axial but not for peripheral manifestations. We evaluated the impact of imaging findings for identification and treatment decisions in patients with peripheral spondyloarthritis (pSpA) and controls (non-SpA). Methods Patients with pSpA (Assessment of SpA international Society criteria, n=30) and non-SpA (n=30), aged

Objective Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA-restricted manner. Methods Human HLA–B27+ T cell lines, specific for the Epstein-Barr virus–derived peptide EBNA258–266, and autologous chondrocytes, serving as nonprofessional antigen-presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy. Results When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA258–266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon-γ production in CD8+ T cells. In mixed chondrocyte–T cell cultures, cell–cell contacts were dependent on the presence of the EBNA258–266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen-specific lysis of target cells. Conclusion Antigen presentation through human chondrocytes allows the stimulation of peptide-specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon-γ–triggered autocrine loop of immune cell–mediated chondrocyte activation in the already inflamed environment. Thus, local HLA-dependent activation of peptide-specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides.

Ankylosing spondylitis (AS) is characterized by two key pathologic findings: sacroiliac joint and spinal inflammation and new bone formation with possible bone fusion, usually in the axial skeleton. Thus, structural damage in AS must be viewed differently than that in rheumatoid arthritis. Tumor necrosis factor blockers effectively inhibit inflammation, as shown by signs and symptoms, function, C-reactive protein, and MRI, and will probably prevent erosive structural damage. However, the ossification of already-damaged bone cannot be influenced by tumor necrosis factor blockade, because these drugs do not inhibit osteoblasts. It remains to be seen whether additional targeting of new bone formation is clinically meaningful in advanced AS. The most important action to prevent structural damage is probably an early diagnosis and effective anti-inflammatory treatment of AS.

Histomorphological analysis shows at least two different patterns in patients with ankylosing spondylitis. Bone marrow edema, lymphocytic infiltrates, increased osteoclast density and increased microvessel density are typical findings in acute inflammation. In areas of new bone formation newly formed trabecular bone shows formation of fibrous tissue in the bone marrow with new cartilage formation and persistently high microvessel density. Morphological changes reminiscent of endochondral ossification can also be observed. Compared to peripheral joints, such as the knee, synovitis is not a predominant finding in the spine, the sacroiliac joints and the hip. Enthesitis is characterised by lymphocytic infiltrates around fibrous cartilage followed by subsequent ankylosis. The molecular mechanisms which promote the transition from inflammation to new bone formation in patients with ankylosing spondylitis are not well understood.

We previously suggested that fibroblast-rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone-forming capabilities, which we approached by searching for bone-forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies.The facet joints of AS patients, osteoarthritis (OA) patients, and autopsy subjects (controls) were screened for subchondral granulation tissue. We searched for hypertrophic chondrocytes by assessing RUNX-2, type X collagen, and matrix metalloproteinase 13 (MMP-13) expression, for osteoblasts by analyzing RUNX-2, CD56, and type I collagen expression, as well as for signs of new bone formation. Adipocytes and lipid accumulation were assessed in Safranin O-stained sections.In the joints of AS and OA patients, RUNX-2-positive cells were found to be lining the granulation tissue. These cells coexpressed type I collagen but lacked type X collagen and MMP-13 expression, confirming their osteoblastic nature. In 91% of AS joints and in 20% of OA joints (P 0.05), we observed foci of new bone formation at contact zones between the granulation tissue and the cartilage. Joints containing bony spots showed greater replacement of the adjacent bone marrow by granulation tissue than did joints without bone formation (P 0.05). The granulation tissue often contained adipocytes and lipid accumulations. Replacement of the subchondral bone marrow by fat tissue was also frequently found but was not associated with new bone formation.The subchondral granulation tissue carries osteoblasts, which promote new bone formation, leading to intraarticular ankylosis of the facet joints in AS.

Background: CD4+ T cell responses to the G1 domain of aggrecan in patients with ankylosing spondylitis (AS) were recently reported. Whether such an immune response can be seen in the CD8+ subpopulation has not yet been determined. Objective: To determine if HLA-B27 restricted G1-specific CD8+ T cells are present in AS and to analyse immunodominant CD8+ T cell epitopes. Methods: Peripheral blood mononuclear cells of 45 patients with AS were stimulated with overlapping 18-mer peptides covering the whole G1 protein. Results were compared with those for patients with rheumatoid arthritis (RA) and healthy controls. For epitope analysis, G1-specific interferon gamma positive (IFNγ+) T cells were isolated by magnetic activated cell sorting. After in vitro expansion, CD8+ T cells were restimulated with 14 subpools of G1 peptides. T cells responding to G1 peptide subpools were quantified by flow cytometry according to IFNγ secretion. Predicted peptides were subsequently confirmed by stimulation with single peptides. Results: G1-specific CD8+ T cell responses were found in 29/45 (64%) patients with AS, 18/35 (51%) patients with RA, but not in healthy controls. Five CD8+ T cell epitopes were identified as immunodominant in five patients. However, the T cell response was not HLA-B27 restricted. Nonamer peptides with an HLA-B27 binding motif did not induce a T cell response. Conclusion: A G1 peptide-specific CD8+ T cell response is present in AS but also in patients with RA. It does not seem to be HLA-B27 restricted. Whether such a response has a role in the pathogenesis of AS needs clarification.

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.

Although non-obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self-Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self-peptides that bind with low affinity to NOD I-A(g7) MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic beta cells. We identified LACK peptides that were the targets of T cells in LACK-immunized NOD mice while binding to I-A(g7) with low affinity. While CD4(+) T cells from NOD mice secreted IFN-gamma, IL-4, IL-5 and IL-10 in response to LACK, those from LACK-expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK-expressing Tg mice exhibited self-reactive CD4(+) T cells with impaired proliferation capabilities. Hence, even self-peptides that bind to I-A(g7) with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.

Activation of autoreactive T cells is critical for the induction of autoimmunity. Several mechanisms may be responsible for the activation of autoreactive T cells in autoimmune diseases. These include cross-reactive microbial peptides (molecular mimicry), viral or bacterial superantigens, release of autoantigen during inflammation, or bystander activation. The molecular mimicry hypothesis postulates that there is significant cross-reactivity between viral and bacterial T-cell epitopes and human self-peptides and that such cross-reactivity can initiate an autoimmune process. At first sight T-cell receptor (TCR) recognition appears to be exquisitely specific since even minor substitutions in a T-cell epitope can diminish or abrogate T-cell activation. The structure of HLA-DR2 with the bound myelin basic protein (MBP) peptide (residues 85 to 99) was determined by X-ray crystallography as a step toward defining molecular mimicry at a structural level. For this purpose, HLA-DR2 was expressed as a soluble protein in the baculovirus system. In the crystal structures of MHC class I-peptide complexes, the P5 side chain occupies a central pocket created by the CDR3 loops of α β TCRs. The dEV8 self-peptide represents a ligand with a low affinity for the 2C TCR. The diverse nature of the molecular mimicry peptides and the ubiquitous presence of some of these pathogens may make it difficult to establish a direct epidemiological link between infectious agents and the occurrence of certain autoimmune diseases.

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

Involvement of the axial skeleton is a characteristic feature of spondyloarthritis (SpA). Asymmetrical arthritis and enthesitis of the peripheral joints are found in about 30–60% of patients.1–3 A 52-year-old patient with a history of psoriasis reported inflammatory back pain, painful ankles and knees and severe morning stiffness on admission. The physical examination revealed tender entheses at different locations: upper thoracic spine, lumbar spinous processes, both iliac crests and posterior superior iliac spines. Furthermore, there was tenderness of both sacroiliac joints, both knees and both ankles without joint swelling. HLA-B27 antigen was negative. The sedimentation rate (70/76) and levels of C reactive protein (1.48 mg/dl) were increased. The Bath Ankylosing Spondylitis Activity Index was 6.0, and the Bath Ankylosing Spondylitis Functional Index was 4.6. Radiographs of the pelvis showed bilateral grade 3 sacroiliitis. Thus, ankylosing spondylitis (AS) associated with psoriasis was diagnosed. Treatment on admission comprised methotrexate (15 mg/week) and prednisolone (5 mg/day). Whole-body MRI (WB-MRI) was performed on a special 1.5 T scanner (Avanto, Siemens, Germany) for evaluation of the extent of suspected widespread inflammatory lesions. Images of the entire body, excluding the hands, were acquired in a head-to-toe scan using parallel imaging and a whole-body surface coil …

Objective In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components, which are mostly unknown. This study was performed to investigate the synovial proliferative T cell response to a panel of recombinant Yersinia antigens in patients with Yersinia ReA and in controls. Methods Synovial fluid mononuclear cells (SFMC) were obtained from 4 patients with Yersinia ReA and from 14 patients with arthritides of different etiology. SFMC were stimulated with 5 recombinant Yersinia antigens (the 19-kd urease β subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and Y enterocolitica heat-shock protein 60 [hsp60]), and with human, Chlamydia trachomatis, and Borrelia burdorferi hsp60. Three T cell clones specific for Y enterocolitica hsp60 were generated from 1 patient with Yersinia ReA. Antigen-induced cytokine release was measured by enzymelinked immunosorbent assay. Results SFMC from all 4 patients with Yersinia ReA responded to each of the Yersinia antigens except the 13-kd protein. These antigens were also recognized by SFMC from a subgroup of patients with undifferentiated arthritis (n = 4), but not by SFMC from other patients with arthritis of different etiology (n = 10). Y enterocolitica hsp60 induced the strongest proliferative response in all cases. Two types of hsp60-reactive T cell clones could be obtained. One clone responded to all hsp60 variants, including the human variant, and showed a type 2 T helper (Th2)-like cytokine-secretion pattern. In contrast, another clone with specificity for the bacterial hsp60 proteins, but not the human equivalent, reacted with a more Th1-like pattern. Conclusion In Y enterocolitica-triggered ReA, at least 4 immunodominant T cell antigens exist, which might be used in lymphocyte proliferation assays to identify patients with Yersinia ReA. The hsp60 is a strong antigen, inducing both bacteria-specific and potentially autoreactive CD4+ T cells of both the Th1 and Th2 type.

Die 63-jahrige Patientin entwickelte uber zwei Jahre langsam progrediente, maximal handflachengrose schmerzhafte Ulzera an beiden Unterschenkeln und eine Nekrose an der rechten Groszehe ohne begleitende Claudicatio. Angiographisch wurden umschriebene Stenosen mittelgroser Gefase, perlschnurartig deformierte Gefassegmente, Gefasrarefizierung in der Peripherie und Verschlusse nachgewiesen. In Anbetracht eines jahrzehntelangen Nikotinabusus (40 Packungsjahre) wurde die Diagnose einer arteriellen Verschluskrankheit (AVK) gestellt. Trotz mehrfacher perkutaner transluminaler Angioplastie (PTA) der A. iliaca communis rechts und A. femoris superficialis verlief die Erkrankung progredient. Im Rahmen der rheumatologischen Konsiliaruntersuchung wurde ein langjahriges Raynaud-Syndrom der Hande erfragt und eine kleine akrale Nekrose am 4. Finger bei beginnender Sklerodaktylie sowie eine dezente Mikrostomie, aber keine diffuse Hautverdickung festgestellt. Bei der kapillaroskopischen Untersuchung fanden sich Megakapillaren und Gefasrarefizierung. Im Serum wurde ein hoher Autoantikorper-Titer gegen Zentrosomen (1:10240) nachgewiesen. Die Assoziation eines solchen klinischen Bildes mit Antikorpern gegen Zentrosomen ist bisher nicht beschrieben worden. Das Gesamtbild wurde als Vaskulopathie bei limitierter systemischer Sklerose und AVK interpretiert. Die Behandlung mit dem Prostacyclinderivat Ilomedin (Iloprost, 50μg i.v./Tag) in Kombination mit einer Endoxan-Pulstherapie (800mg i.v./Monat) fuhrte zu deutlicher subjektiver Besserung, weitgehender Abheilung der Ulcera und deutlicher Reduktion des Schmerzmittelverbrauchs.

To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS).Histomorphologic and histomorphometric analyses were performed on zygapophyseal joints obtained from 18 patients with AS, 9 patients with osteoarthritis (OA), and 10 cadaver donors without a rheumatic disease (controls). The proteoglycan content of the cartilage was determined by Safranin O staining and the chondrocyte apoptosis according to caspase 3 expression.AS joints were categorized into 3 groups according to the morphology of the joint surfaces and joint space: group 1 were joints with an open joint space, group 2 were joints with cartilaginous fusion, and group 3 were joints with bony fusion of the joint surfaces. Progressive loss of the joint space from group 1 joints to group 3 joints suggests that this grouping corresponds to sequential stages of joint remodeling. Cartilage thickness and subchondral bone plate thickness declined from group 1 to group 3 (P0.01). Increased chondrocyte apoptosis rates were found in groups 1 and 2 (P0.05), while in group 3, a reduction in the proteoglycan content was found (P0.001). Bone marrow replacement and invasion of the subchondral bone plate by fibrous tissue was found predominantly in AS joints in group 2.Cartilage degeneration, indicated by cartilage thinning, enhanced chondrocyte apoptosis, and proteoglycan loss, and subchondral bone thinning, promoted by invasion of the subchondral bone plate by a fibrous tissue originating from the bone marrow, are hallmarks of joint remodeling in AS.

Introduction: The molecular mechanisms of syndesmophyte formation in ankylosing spondylitis (AS) are yet to be characterised. Molecules involved in bone formation such as Wnt proteins and their antagonists probably drive syndesmophyte formation in AS. Methods: This study investigated sequential serum levels of functional dickkopf-1 (Dkk1), a potent Wnt antagonist involved in bone formation in arthritis, by capture ELISA with its receptor LRP6 in 65 AS patients from the German Spondyloarthritis Inception Cohort. Dkk1 levels were then related to structural progression (syndesmophyte formation) as well as sclerostin and C-reactive protein (CRP) levels. Results: Functional Dkk1 levels were significantly (p=0.025) higher in patients with no syndesmophyte growth (6.78±5.48 pg/ml) compared with those with syndesmophyte growth (4.13±2.10 pg/ml). Dkk1 levels were highly correlated to serum sclerostin levels (r=0.71, 95% CI 0.53 to 0.82; p

Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease β-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease β-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.

Objective.Regulatory T cells are characterized by expression of the transcription factor FoxP3 and are thought to be involved in the pathogenesis of autoimmune diseases. We determined the frequency and phenotypic characteristics of CD4+FoxP3+ T cells in the blood and synovial fluid (SF) of patients with inflammatory joint diseases.Methods.SF from 10 patients with ankylosing spondylitis (AS), 20 patients with other spondyloarthritides or with peripheral arthritis (pSpA), and 12 patients with rheumatoid arthritis (RA), and peripheral blood (PB) from 22 patients with AS, 19 with pSpA, 15 with RA, and 12 healthy controls were stained for CD4, FoxP3, CD25, and CD127 and different effector cytokines and then analyzed by flow cytometry. Methylation pattern of the Treg-specific demethylated region (TSDR) was determined after bisulfite treatment by quantitative polymerase chain reaction.Results.In all groups of patients we observed higher frequencies of Foxp3+ cells/CD4+ T cells within SF compared to PB. The frequency of synovial Foxp3+ cells/CD4+ T cells was significantly higher in patients with pSpA (18.79% ± 6.41%) compared to patients with AS (9.69% ± 4.11%) and patients with RA (5.95% ± 2.21%). CD4+FoxP3+ T cells were CD25+ and CD127− and lacked effector cytokine production in any of the different patient groups. The majority of the CD4+CD25+CD127− T cells showed demethylation of the TSDR within the Foxp3 locus, confirming its regulatory phenotype.Conclusion.Our data show accumulation of Foxp3+ T cells within inflamed joints. These Foxp3+ T cells are mainly of stable T regulatory phenotype. The high frequency of Foxp3+ T cells in pSpA might contribute to the spontaneous resolution and remitting course of arthritis in pSpA as compared to the more persistent joint inflammation in RA.

The principal clinical and radiological feature of all axial spondyloarthritis is an involvement of the axial skeleton (sacroiliac joints and spine) and, to a lesser extent, the hip joints. Immunohistochemical studies provide worthwhile information regarding disease mechanisms in axial spondyloarthritis. Immunohistochemical investigation of sacroiliac joints, spine and hip joints suggested an important role of T-cells in the development of acute inflammatory lesions. Furthermore, activated angiogenesis and macrophage/osteoclast activation also play a relevant role in the development of early active inflammatory lesions. Nonetheless, the mechanisms leading to activation of osteoproliferation with subsequent syndesmophyte and ankylosis formation in patients with spondyloarthritis remains unclear. It is suggested that the Wnt pathway is actively involved in this process due to decreased expression of new bone formation inhibitors such as sclerostin. However, to understand the full picture of the interrelationship between inflammation and new bone formation, and to explore new treatment targets for suppression of the excessive bone formation as well, further investigations are needed.

Objective Osteocytes are considered to be sensors of bone damage and regulators of bone mass by specifically expressing sclerostin, an inhibitor of bone formation. The contribution of osteocytes in regulating local bone remodeling in arthritis is unknown. The aim of this study was to investigate the role of osteocytes as contributors to bone remodeling in ankylosing spondylitis (AS). Methods Sclerostin expression and osteocyte death were assessed by immunohistochemistry in joints derived from patients with AS, patients with rheumatoid arthritis (RA), and patients with osteoarthritis (OA), as well as from control subjects. In addition, the serum level of sclerostin was assessed by enzyme-linked immunosorbent assay in healthy subjects and patients with AS; this assessment included the longitudinal correlation of sclerostin serum levels and radiographic progression in the spine of patients with AS. Results Sclerostin expression was confined exclusively to osteocytes. Whereas the majority of osteocytes in healthy individuals and patients with RA were sclerostin positive, expression was significantly reduced in patients with OA and was virtually absent in patients with AS. Moreover, serum levels of sclerostin were significantly lower in patients with AS than in healthy individuals. Importantly, low serum sclerostin levels in patients with AS were significantly associated with the formation of new syndesmophytes (P = 0.007). Conclusion Sclerostin expression is impaired in patients with AS, suggesting a specific alteration of osteocyte function in this disease. A low serum level of sclerostin in the setting of AS is linked to increased structural damage, emphasizing the role of sclerostin in the suppression of bone formation.

In light of the impressive efficacy of tumor necrosis factor blockers in the treatment of ankylosing spondylitis, particularly in patients with short disease duration, defining outcome parameters to monitor the structural damage of the disease has become more pertinent. In this Viewpoint the authors explore the relevance of osteoproliferation amongst other outcome parameters.

We describe 3 AS patients treated with etanercept for active AS who developed new onset of CD while AS related symptoms responded well to etanercept. Typical symptoms of active CD occurred 11, 12, and 26 months after start of etanercept therapy, respectively. On colonoscopy, inflammatory lesions highly compatible with CD were found endoscopically and histologically in all patients. Etanercept was stopped, and CD responded well to standard treatment. One of the 3 patients was reexposed to etanercept later on. While the 2 patients without re-exposure to etanercept did not have further CD flares, the third patient flared 6 months after re-institution of etanercept in combination with azathioprine. New onset of CD may be considered as an immune-mediated injury induced by etanercept. Although this is an intriguing hypothesis, any causative role of etanercept remains unproven at this stage.

Anti-tumor necrosis factor-alpha (TNF-alpha) therapy can induce reactivation of tuberculosis and an increase of other infections in patients with ankylosing spondylitis (AS). This raises the question if an alteration of T cell function can be detected by in vitro analysis to identify patients who might be more at risk of acquiring such infectious diseases.We examined peripheral blood from AS patients without history of tuberculosis before and after 10-14 and 24-36 weeks of therapy with adalimumab (n = 8) or infliximab (n = 10). Fresh peripheral blood mononuclear cells were stimulated with cytomegalovirus antigens and with the Mycobacterium tuberculosis antigen purified protein derivative and early secretory antigen target 6. Interferon-gamma production of CD4+ T cells was assessed after in vitro antigen-specific stimulation by intracellular cytokine staining and flow cytometry.There was no significant change, either decrease or increase, of the T cell response to recall antigens during therapy compared to controls without treatment, if the mean values of all patients treated with adalimumab or infliximab were compared at the given timepoints. However, analysis on the individual patient level of such T cell responses revealed 1 adalimumab-treated patient and 2 infliximab-treated patients with a clear decrease of T cell response during therapy. Longterm analysis indicated that such a decrease of T cell responsiveness is generally transient and reconstituted at the latest after 52 weeks.Some patients treated with adalimumab or infliximab showed a decrease of T cell responsiveness, which seems to be transient. These patients in particular might be at risk for intracellular infections.

To the Editor: We thank Pacheco-Tena, et al 1 for their letter. … Address correspondence to Dr. H. Appel, Charite Universitatsmedizin Berlin, Campus Benjamin Franklin, Rheumatology, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: heiner.appel{at}charite.de

Background Ankylosing spondylitis (AS) is characterized by erosive bone damage followed by bone formation. A recent MRI study suggested that ankylosis of sacroiliac joints develops following repair of erosions with fat metaplasia and backfill as intermediary steps in this pathway [1]. Objectives In this study we used histological analysis of facet joints of AS patients to elucidate the mechanism of bone formation and the potential role of adipogenic tissue transformation. Methods Sections of facet joints which can also undergo ankylosis in AS were used from patients with AS, patients with osteoarthritis (OA) and from controls (CO). In safranin O stained tissue sections, the subchondral bone marrow area and sites of erosions were evaluated with regard to replacement by fibrous tissue, a pathological feature described before, and adipogenic tissue transformation. To identify putative bone forming cells we analyzed the expression of Runx2 a shared marker of chondrocytes and osteoblasts, and collagen type X and MMP13 as specific markers of hypertrophic chondrocytes as well as CD56 and collagen 1 as markers of osteoblasts within eroded areas of the subchondral endplate and the articular cartilage. Results Within the subchondral bone marrow adipogenic tissue transformation as well as fibrous transformation was found: 10/16 (62.5%) of CO joints, 18/29 (62%) of AS joints and 16/17 (94%) of OA joints showed replacement of bone marrow by fat marrow of various extend (up 100% of the bone marrow area/slide) while 0/16 (0%) of control joints, 12/29 (41.4%) of AS joints and 14/17 (82%) of OA joints showed transformation into fibrous tissue. Among AS joints, replacement of bone marrow by fibrous tissue was found more often in joints with cartilaginous fusion (5/6) compared to joints with bony fusion (4/15, p 0.05). At sites of subchondral bone and cartilage erosion we always found fibrous tissue. Runx2+ cells were detectable at the borders of fibrous tissue in AS and OA joints. The lack of collagen type X and MMP13 expression but osteocalcin and collagen type I expression of these cells proved the osteoblastic nature of these cells. Formation of new bone was found at contact areas between fibrous tissue and eroded articular cartilage – a phenomenon occurring more often in AS joints than in OA joint, i.e. in 92% of AS joints and 27% of OA joints (p Conclusions In AS, osteoblasts lining the fibrous tissue facilitate bone formation at sites of bone and cartilage erosions which promotes joint ankylosis. Fat marrow is rather found within the adjacent subchondral bone marrow. References Maksymowych WP, Wichuk S, Chiowchanwisawakit P, Lambert RG, Pedersen SJ: Fat metaplasia and backfill are key intermediaries in the development of sacroiliac joint ankylosis in patients with ankylosing spondylitis. Arthritis Rheumatol 2014, 66(11):2958-2967. Disclosure of Interest None declared

Objective Zygapophyseal joints of the spine are often affected in ankylosing spondylitis (AS). In this study, we undertook a systematic immunohistologic evaluation of the immunopathology of the zygapophyseal joints in patients with advanced AS. Methods We obtained zygapophyseal joints from 16 AS patients undergoing polysegmental correction of kyphosis and from 10 non-AS controls (at autopsy). Immunohistologic analysis of the bone marrow was performed by analyzing the number of infiltrating T cells (CD3, CD4, CD8), B cells (CD20), osteoclasts (CD68), bone marrow macrophages (CD68), and microvessel density (CD34) per high-power field. Results Zygapophyseal joints from 6 of 16 AS patients, but from none of the controls, exhibited 2 or more CD3+ T cell aggregates, signifying persistent inflammation. Interstitial CD4+ and CD8+ T cells were significantly more frequent in AS patients compared with non-AS controls (P = 0.002 and P = 0.049, respectively). While there was no clear difference between the number of CD20+ B cells in AS patients overall compared with controls, there was a significant difference when persistently inflamed joints from patients with AS were compared with joints without active inflammation from patients with AS or joints from controls (both P = 0.03). Microvessel density in bone marrow from AS patients with active inflammation was significantly higher than that in bone marrow from controls. Conclusion This immunohistologic study of bone marrow from zygapophyseal joints demonstrates persistent inflammation in the spine of patients with AS, including those with longstanding disease. The findings of increased numbers of T cells and B cells and neoangiogenesis suggest that these features play a role in the pathogenesis of AS.

Objective Previous histopathologic and magnetic resonance imaging studies suggest that the subchondral bone marrow might be the primary site of inflammation in patients with ankylosing spondylitis (AS) and that this might be reflected by inflammation found in hip joints. The aim of this study was to conduct an immunohistologic assessment of the bone–cartilage interface and subchondral bone marrow in AS patients with hip arthritis. Methods We collected femoral heads from patients with AS, osteoarthritis (OA), and rheumatoid arthritis (RA) who were undergoing hip replacement. The subchondral bone marrow and bone–cartilage interface were assessed immunohistochemically by evaluating infiltrating T cells, microvessel density, and osteoclasts. Areas of the femoral head surface with and without cartilage were assessed separately. Results At sites with surface cartilage, we found subchondral infiltration of CD3+ T cell aggregates at significantly higher numbers in AS patients as compared with OA patients, but not RA patients. At sites of complete cartilage destruction, the frequency of CD3+ T cell aggregates was significantly reduced as compared with sites with cartilage on the surface in AS patients, but not in RA patients. Similar differences were found for CD4+ and CD8+ T cells. Only at sites with surface cartilage, but not those without, angiogenesis and osteoclastic foci in the subchondral bone marrow in AS patients were significantly increased as compared with RA patients and with OA patients. Conclusion These findings suggest that the subchondral bone marrow and bone–cartilage interface are primary sites of inflammation in AS and that cartilage might be necessary for the induction of inflammation.

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

Objective Several hypotheses have been proposed to explain the strong association between HLA–B27 and ankylosing spondylitis (AS). Among these, the arthritogenic peptide theory proposes that certain B27 subtype alleles bind specific arthritogenic peptide(s) due to their unique amino acid anchor residues. Cartilage antigens have been discussed as candidate targets for the immune response in AS. The recognition of HLA–B27–peptide complexes by self-reactive CD8+ T cells might contribute to joint-specific tissue damage. Therefore, we investigated the presence of autoreactive CD8+ T cells specific for cartilage-derived peptides in patients with AS. Methods An HLA–B27–binding prediction program and a proteasome-cutting prediction program for the human 20S proteasome were used to screen 18 human cartilage proteins for potentially immunogenic nonamer peptides. The peptides identified were used to stimulate peripheral blood mononuclear cells from 20 HLA–B27–positive patients with AS and synovial fluid (SF) mononuclear cells from 7 HLA–B27–positive patients with AS. Activation of T cells was measured by antigen-specific intracellular cytokine staining and quantified by flow cytometry. Results From the screening analysis, we identified 121 nonamer peptides. Of these, 1 peptide derived from type II collagen and 1 from type VI collagen were stimulatory for peripheral blood CD8+ T cells in only 1 of 20 patients. However, in 4 of 7 SF samples the same type VI collagen–derived nonamer peptide stimulated SF CD8+ T cells, but none of the other peptides was stimulatory. This CD8+ T cell response could be blocked by an anti–HLA–B27 antibody, confirming an HLA–B27–restricted immune response. Conclusion Our findings suggest that cartilage-directed cellular autoimmunity might play an important role in joint-specific tissue damage in patients with AS. Future research is necessary to determine whether the identified peptide is of pathogenetic relevance.

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Congenital absence of portal vein (CAPV) with a systemic shunt of enteric blood is a rare malformation. Twenty-seven cases have been reported to date; with the exception of 4 patients all cases had either liver anomalies or cardiac anomalies. CAPV is usually diagnosed before the age of 18 (25/27 cases). Here we present the case of a 33-year-old woman with CAPV without further anomalies. Beside suffering from circumscript scleroderma, splenomegaly and hypersplenism, the patient was otherwise healthy. Diagnosis was based on histological findings, angiography and CT scan. The liver biopsy showed portal tracts with inconspicuous arteries and bile ducts, but with severe loss of portal vein branches and minimal proliferation of bile ductules. In addition, the perivenular sinusoids were slightly dilated with focal parenchymal atrophy. Angiography showed a missing portal vein system and a missing splenic vein with drainage of the enteric blood through dilated perisplenic and perigastric collaterals to the azygos vein system. A CT scan revealed an empty liver hilus without portal vein. Here we discuss clinical aspects of this patient, show radiologic and histopathologic findings, and compare them to other cases in the literature.

Reactive arthritis (ReA) mostly presents as an asymmetrical oligoarthritis, usually affecting the leg joints.1 It is a T cell dependent inflammatory disease following infections with various enteropathic bacteria—for example, Yersinia enterocolitica 0:3 , Salmonella enteritidis, Shigella fexneri, or microbes pathogenic for the urogenital tract such as Chlamydia trachomatis. 2 Identification of the structure of antigens of triggering microbes which drive immune responses in the synovial fluid would be of great interest. We recently described two major antigens, the Ye 19 kDa and the Ye HSP60 inducing synovial T cell proliferation in patients with Ye triggered ReA.3–7 They seemed to overcome the specificity problem of proliferation assays with T cells cultured from the site of inflammation to ReA triggering bacteria, because the local immune response to whole bacteria …

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for patients with ankylosing spondylitis (AS). They are effective in reducing signs and symptoms, and increasing evidence suggests that their sustained use may also retard radiographic progression [1, 2]. The equal efficacy of cyclooxygenase (COX)-2 selective inhibitors in AS suggests that COX-2-dependent PGE 2 synthesis is involved in the pathogenesis of AS. Objectives We studied here the expression of COX-2 and PGE 2 and of the PGE 2 receptors EP 2 and EP 4 in zygapophyseal joints of AS patients. Methods COX-2, PGE 2 , EP 2 and EP 4 were determined by immunohistochemistry in zygapophyseal joints from 19 AS, 11 osteoarthritis patients (OA) and 12 autopsy controls (CO). COX-2 + , PGE + , EP + and EP + cells were detected at four different sites: within hyaline cartilage, subchondral bone plate, fibrous tissue and subchondral bone marrow. Results Within the subchondral bone marrow, numbers of COX-2 + and PGE + cells were higher in joints of AS patients compared to controls (p=0.0394, p=0.0138). The number of cells expressing EP 2 and EP 4 were also but non-significantly elevated in the bone marrow of AS patients (p=0.0630; p=0.0786). In contrast, we found in preliminary analysis very low expression of COX-2 within the cartilage, the subchondral bone and the subchondral fibrous tissue. Within the subchondral bone plate, numbers of PGE + and EP + cells were higher in AS (and OA) patients compared to controls (p Comparably very high numbers of PGE + and EP + cells were detected within the fibrous tissue of AS and OA patients (p=0.1902, p=0.3726). Conclusions According to these data, COX-2 dependent PGE 2 synthesis is primarily seen in the subchondral bone marrow and elevated in AS patients, and might be the target site for drugs inhibiting COX-2. References Poddubnyy D, Rudwaleit M, Haibel H, Listing J, Marker-Hermann E, Zeidler H, et al. Effect of non-steroidal anti-inflammatory drugs on radiographic spinal progression in patients with axial spondyloarthritis: results from the German Spondyloarthritis Inception Cohort. Annals of the rheumatic diseases. 2012 Oct; 71(10):1616-1622. Wanders A, Heijde D, Landewe R, Behier JM, Calin A, Olivieri I, et al. Nonsteroidal antiinflammatory drugs reduce radiographic progression in patients with ankylosing spondylitis: a randomized clinical trial. Arthritis and rheumatism. 2005 Jun; 52(6):1756-1765. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3576