The Association of Fecal Microbiota in Ankylosing Spondylitis Cases with C-Reactive Protein and Erythrocyte Sedimentation Rate.

The purpose of this work was to identify the features of the gut microbiome in cases of ankylosing spondylitis (AS) testing positive for human leukocyte antigen- (HLA-) B27 and healthy controls (HCs) as well as to determine how bacterial populations were correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Fecal DNA extracted from fecal samples from 10 AS cases and 12 HCs was subjected to 16S rRNA gene sequencing. The two research groups did not differ significantly regarding alpha diversity. By comparison to HCs, AS cases displayed a lower relative level of Bacteroidetes (P < 0.05), but a higher level of Firmicutes and Verrucomicrobia (P < 0.05). Furthermore, the correlation between the specific gut bacteria and ESR or CRP was investigated. At the phylum level, Firmicutes and Verrucomicrobia had a positive association with ESR and CRP, while Bacteroidetes exhibited an inverse correlation with ESR and CRP. Meanwhile, in terms of genus, Bacteroides had a positive association with ESR and CRP, whereas Ruminococcus and Parasutterella had an inverse correlation with ESR and CRP, and Helicobacter also displayed an inverse correlation with CRP. Such findings indicated dissimilarities between AS cases and HCs regarding the gut microbiome, as well as the existence of correlations between bacterial populations and both ESR and CRP.

HLA-B27 alters BMP/TGFß signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis.

OBJECTIVES: The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. METHODS: We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human ß2-microglobulin (hß2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA. RESULTS: Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hß2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor ß (TGFß). CONCLUSIONS: Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFß/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.

HLA superfamily assignment is a predictor of immune response to cancer testis antigens and survival in ovarian cancer.

OBJECTIVES: To characterize the association between major histocompatibility complex (MHC) types and spontaneous antibody development to the cancer testis (CT) antigen NY-ESO-1. METHODS: Tumor expression of NY-ESO-1 and serum antibodies to NY-ESO-1 were characterized in addition to human leukocyte antigen (HLA) type for patients with epithelial ovarian cancer. HLA types were assigned to structure-based superfamilies and statistical associations were examined. HLA types were compared to existing reference libraries of HLA frequencies in a European-Caucasian American population. RESULTS: Out of 126 patients identified, 81% were expression positive and 48% had spontaneous antibody responses to NY-ESO-1. There was an association between HLA-B superfamily and seropositivity among patients with tumors expressing NY-ESO-1 (p<0.001). The differences in HLA-B superfamily assignment were driven by HLA-B44. Among all patients, the B27 superfamily was over-represented compared with the general population (p<0.001). CONCLUSIONS: HLA type appears to be associated with spontaneous anti-CT antigen antibodies, as well as with the overall risk of ovarian cancer.

Altered bone material properties in HLA-B27 rats include reduced mineral to matrix ratio and altered collagen cross-links.

Spondyloarthropathy and inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, are often associated with severe osteopenia/osteoporosis in both children and adults. HLA-B27 transgenic rats present a phenotype that includes severe colitis and severely accelerated alveolar bone loss. The purpose of this study was to evaluate long bone density status, systemic bone metabolic markers, and intrinsic bone material properties in HLA-B27 transgenic (TG) rats, and compare them with those of age- and sex-matched wild-type (WT) animals. The results indicate that in the HLA-B27 rat, an animal susceptible to both alveolar bone loss (ABL) and long bone osteopenia, there is a statistically significant negative correlation between ABL and long bone bone mineral density (BMD), as well as mineral/matrix ratio at active bone-forming trabecular surfaces. The TG animals had a lower mineral/matrix ratio and higher relative proteoglycan and advanced glycation end product (ϵ-N-Carboxymethyl-L-lysine) content and pyridinoline/divalent collagen cross-link ratio compared with WT. These results may provide better understanding of the interrelationship between osteoporosis and oral bone loss, the underlying causes of the inferior bone strength in the HLA-B27 transgenic animals, and could prove to be a useful model in the elucidation of the pathophysiology of spondyloarthropathy and IBD-associated osteopenia/osteoporosis and in the evaluation of pharmacological intervention(s) against such conditions.

The classification and diagnostic criteria of ankylosing spondylitis.

Ankylosing spondylitis is the prototype of immune-mediated inflammatory rheumatic diseases grouped under the term spondyloarthritis (SpA). An early diagnosis has now become increasingly important because effective therapies are available and anti-TNF drugs are even more effective if used in early stages of the disease. In ankylosing spondylitis, the 1984 modified New York criteria have been used widely in clinical studies and daily practice but are not applicable in early disease when the characteristic radiographic signs of sacroiliitis are not visible but active sacroiliitis is readily detectable by magnetic resonance imaging (MRI). Thus there has been a need for new classification or diagnostic criteria to identify inflammatory spondyloarthritis at early stage of the disease. This led to the concept of axial SpA to include the entire spectrum of patients with axial disease both, with and without radiographic damage. New classification criteria for the wider group of SpA have been proposed by ASAS (Assessment of Spondylo Arthritis International Society); and the patients are sub-grouped into (1) a predominantly axial disease, termed axial SpA including AS and non-radiographic axial SpA; (2) peripheral SpA. The clinical course and disease process of non-radiographic axial spondyloarthritis remains unclear. However the development of the SpA criteria by ASAS particularly for axial SpA, is an important step for early diagnosis and better management of these patients.

Allicin attenuates inflammation and suppresses HLA-B27 protein expression in ankylosing spondylitis mice.

Here we aimed to determine the therapeutic effect of allicin on ankylosing spondylitis (AS) and explore the mechanism(s) of action. AS mouse model was constructed by transferring the HLA-B2704 gene into Kunming mice and verified by RT-PCR and CT imaging. Verified AS mice were randomly divided into model group (n = 6) and allicin-treated groups (50, 100, and 200 mg/kg, resp., n = 6, p.o., for 2 months). Wild type mice were used as control (n = 6). The levels of AS-related inflammatory factors were measured by ELISA. mRNA and protein expressions of HLA-B27 were checked by RT-PCR and western blotting. As the results, the mouse model of AS was successfully established, and high-dose allicin could markedly alleviate spine inflammatory injury possibly via reducing the secretion of the inflammatory factors (IL-6, IL-8, and TNF- α ) sharply in AS mice. Moreover, allicin significantly inhibited HLA-B27 protein translation but failed to suppress HLA-B27 gene transcription in AS mice, indicating a posttranscriptional mechanism of this modulation. In conclusion, allicin has potential to be used for AS treatment as an anti-inflammatory nutraceutical.

Co-expression of HLA-B7 and HLA-B27 alleles is associated with B7-restricted immunodominant responses following influenza infection.

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vß8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vß12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens.

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 monochain transgenic/H-2 class I null mice: novel versatile preclinical models of human T cell responses.

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human ß2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ-producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell-based vaccines.

The expression of human leukocyte antigen G (HLA-G) is associated with sacroiliitis stages of ankylosing spondylitis.

Recent studies have demonstrated that human leukocyte antigen G (HLA-G) may play an important role in autoimmune diseases. The present study is to investigate whether or not HLA-G is associated with sacroiliitis stages of ankylosing spondylitis (AS), a systemic autoimmune disease. Plasma levels of soluble HLA-G (sHLA-G) and HLA-G expression on the surface of peripheral blood mononuclear cells (PBMCs) were measured in 55 AS patients and 49 healthy controls by using a specific HLA-G ELISA and flow cytometric (FCM) analysis, respectively. Association of HLA-G expression with sacroiliitis stages of the patients was statistically analyzed. The plasma sHLA-G concentrations were noticeably lower in the AS patients when compared to the healthy controls while the mean fluorescence intensity (MFI) of the HLA-G expression on the surface of PBMCs was significantly higher in the AS patients than in the healthy controls (both P<0.0001). The HLA-G expression on the surface of PBMCs, plasma sHLA-G levels and HLA-B27 expression were significantly correlated to each other. Moreover, the plasma sHLA-G was inversely associated with the sacroiliitis stages (P=0.008), while the HLA-G expression on the surface of PBMCs increased from stage 0 to II but decreased in stage III (P=0.001). The significant association of HLA-G expressions with AS sacroiliitis stages suggests that HLA-G is possibly involved in the pathology of the disease. The detection of HLA-G expression may therefore be a useful laboratory test to reveal disease process in AS patients.

Early recognition of aortitis of the aorta ascendens with ¹8F-FDG PET/CT: syphilitic?

We present the case of a 42-year-old woman known with a human leukocyte antigen B27 positive ankylosing spondylitis. Despite treatment with a tumor necrosis factor blocking agent, the patient was not pain free and inflammation markers remained elevated. An (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) was performed in an attempt to exclude possible other inflammatory processes. The (18)F-FDG PET/CT revealed increased metabolic activity in the ascending aortic wall, which appeared unexpectedly related to late syphilis. Based on this case and existing literature on this subject, we come to the conclusion that (18)F-FDG PET/CT can help in an early establishment of syphilitic aortitis before the possible life-threatening sequelae of syphilitic aortitis occur.

Genetic polymorphisms of stromal interaction molecule 1 associated with the erythrocyte sedimentation rate and C-reactive protein in HLA-B27 positive ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The development of ankylosing spondylitis is still unclear. Genetics factors such as human leukocyte antigen HLA-B27 and ERAP1 have been widely reported to associate to AS susceptibility. In this study, we enrolled 361 AS patients and selected four tagging single nucleotides polymorphisms (tSNPs) at STIM1 gene. The correlation between STIM1 genetic polymorphisms and AS activity index (BASDAI, BASFI, BAS-G) as well as laboratory parameters of inflammation (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)) were tested. Our results indicated that HLA-B27 positive AS patients who are carrying the minor allele homozygous G/G genotype of SNP rs3750996 significantly associated with a higher level of ESR in serum. Furthermore, rs3750996/rs3750994 pairwise allele analysis indicated that G-C haplotypes also significantly correlated with higher level of ESR as well as CRP. These findings provide a better understanding of STIM1 genetic contribution to the pathogenesis of AS.

Th17 cells expressing KIR3DL2+ and responsive to HLA-B27 homodimers are increased in ankylosing spondylitis.

CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B27(2)), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B27(2)-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2(+) CD4 T cells. KIR3DL2(+) CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2(+) cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2(+) CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2(+) lines from controls or KIR3DL2(-) CD4 T cells. Strikingly, KIR3DL2(+) CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.

Association of IL1R polymorphism with HLA-B27 positive in Iranian patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is one of the most common causes of inflammatory arthritis, with an estimated prevalence of 0.1-0.9%. Genetic factors have been strongly implicated in its aetiology, and heritability as assessed by twin studies has been estimated to be >90%. HLA- B27 is almost essential for inheritance of AS; it is not merely sufficient for explaining the pattern of familial recurrence of the disease. This study's purpose is to investigate the association of ankylosing spondylitis with single-nucleotide polymorphisms (SNPs) in the IL-1 family: IL-1a (-889C/T) rs1800587, IL-1b (-511C/T) rs16944, IL-1b (+3962C/T) rs1143634, IL-1R (Pst-1 1970C/T) rs2234650 and IL-1RA (Mspa-1 11100C/T) rs315952. 99 unrelated Iranian AS patients and 217 healthy control subjects were selected. Cytokine typing was performed by the polymerase chain reaction with sequence-specific primers assay. The allele and genotype frequencies of the polymorphisms were determined: The IL1α rs1800587, IL1ß rs16944 and IL1ß rs1143634 were not significantly associated with AS. Genotype frequencies at IL1R rs2234650 differed between cases and controls (χ(2)=8.85; p=0.01); the IL1R rs2234650 C/T and T/T genotypes were less common in AS patients than controls. The IL1R rs2234650 C/T genotype was inversely associated with AS comparing with the IL1R rs2234650 C/C genotype (OR=0.48; p=0.005). IL1R rs2234650 C/T genotype was less common in patients than controls (OR=0.37; p=0.02).Furthermore IL1R rs2234650 T allele was strongly associated with HLA-B2702 patients rather than HLA-B2705 but was not associated with HLA-B27 negative patients (OR=0.33; p=0.01). Polymorphisms of IL1α rs1800587, IL1ß rs16944 and IL1ß rs1143634 were not significantly associated with ankylosing spondylitis but inversely in this study IL1R rs2234650 was significantly associated and carriage of T allele in IL1R rs2234650 seems to be protective, while carriage of C allele result in two fold higher risk of developing AS.

Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the ubiquitin proteasome pathway.

OBJECTIVES: To determine if peripheral blood monocytes from patients with ankylosing spondylitis (AS) differed in protein expression compared to rheumatoid arthritis (RA) and healthy controls (HC). METHODS: Monocyte protein expression was characterised by 2D gel electrophoresis and by label-free quantitative expression profiling, using nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS(E), where (E) refers to low/high collision energy switching). Data sets were analysed using the Waters expression profiling system and Ingenuity pathway analysis (IPA). RESULTS: Two-dimensional gel electrophoresis showed upregulation of proteasomal constituents in AS monocytes, including the beta subunit of proteasome activator (PA)28. Monocyte expression profiling and IPA showed that significant changes in protein expression within the ubiquitin proteasome pathway (UPP) were restricted to AS monocytes. Statistically significant differences in protein expression involving the leucocyte extravasation, vascular endothelial growth factor, integrin and Toll-like receptor signalling pathways were seen in AS and RA monocytes compared to healthy controls. No evidence of upregulation of proteins involved in the endoplasmic reticulum stress response pathway was found in either AS or RA monocytes. Finally, the PA28 complex was shown to increase the generation of human leucocyte antigen (HLA)-B27 antigenic epitopes by the proteasome in vitro. CONCLUSIONS: Our proteomic analyses support the hypothesis that monocytes play an important role in the pathogenesis of AS and RA, and further suggest a specific role in AS for the UPP. Quantitative proteomic expression profiling constitutes a powerful new tool for rheumatology research.

Is HLA-B27 a useful test in the diagnosis of juvenile spondyloarthropathies?

INTRODUCTION: Seronegative spondyloarthritis (SSA) is a type of arthritis that involves joints in the spine, as well as the hips, shoulders, knees and ankles. The diagnosis of juvenile spondyloarthritis is rarely entertained in young children who present with back and leg pain. The aim of the present study was to assess the role of HLA-B27 as a diagnostic marker in children with spondyloarthropathy, and correlation of HLA-B27 with radiological features and tuberculosis. METHODS: Routine haematological and immunological tests were done by standard method and HLA-B27 typing was done by the lymphocytotoxicity method. A total of 70 cases of juvenile spondyloarthropathy were studied from May 2006 to September 2007. It included both males and females. RESULTS: Positivity of HLA-B27 in childhood SSA was only 71.4 percent (50/70). Gender-wise analysis showed that 76.7 percent (46/60) males and 20.0 percent (2/10) of the female patients were HLA-B27 positive. In HLA-B27-positive cases, the sacroiliac, hip, ankle, lower spine and knee joints were more involved. Urinary tract infection, diarrhoea and constipation were more common in HLA-B27-positive cases. None of the HLA-B27 cases were positive for rheumatoid factor; however, C-reactive protein was raised in 60.5 percent. In bilateral/unilateral sacroiliitis diagnosed by radiographs, only 81.5 percent patients were HLA-B27 positive. Tuberculosis was diagnosed in 14.3 percent (10/70) of total cases in which HLA-B27 positivity was seen in 60 percent of cases (6/10). CONCLUSION: Our study concludes that both HLA-B27 and radiological tests should be done in male children suspected to have SSA, because it can indicate early cases of juvenile spondyloarthropathy when radiological changes are not present, and it produces a more severe disease. HLA-B27 positivity probably also predisposes to tuberculosis.

Determination of HLA-B27 subtypes in Iranian patients with ankylosing spondylitis.

The human leukocyte antigen-B27 is one of the class I molecules of the major histocompatibility complex which is strongly associated with ankylosing spondylitis (AS). The strength of the disease association with B27 varies markedly among racial and ethnic populations. It is an allele family, which constitutes about 31 subtypes, with a considerable geographic and ethnic difference in distribution. It is important to know whether certain subtypes show any preferential association with AS. Because there is no report regarding HLA-B27 subtypes in Iranian patients with AS, the main purpose of the present study was to assess the frequency of subtypes of human leukocyte antigen (HLA)-B27 in patients with ankylosing spondylitis in Iranian populationOne hundred and nineteen AS patients (82 HLA-B27 positive and 37 HLA-B27 negative) were selected for this study. HLA-B27 positive patients were screened by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) for B*27 subtyping.The results of present study revealed that only two subtypes were detected in Iranian patients, including B*2705 (52 patients, 63.4%) and B*2702 (30 patients, 36.6%). Our results showed a restricted number of HLA-B27 subtypes associated with AS in Iran and an elevated frequency of the B*2705 allele in these patients similar to other Euro-Caucasoid (Aryan) groups in the world.

Pathogenesis of ankylosing spondylitis: current concepts.

More than three decades after the discovery of HLA-B27 as a major genetic clue to the origins of ankylosing spondylitis, much has been learned about pathogenesis. However, the role of this major histocompatibility complex class I allele remains undefined. Studies from animal models have demonstrated that HLA-B27 overexpression can cause inflammatory disease with spondyloarthritis features, and together with investigations of patient-derived material, both innate adaptive and immune responses have been implicated. The gastrointestinal immune response to pathogens and even normal flora, with subclinical or overt inflammation, may play a role as an environmental component of these diseases. Although there has been a large conceptual emphasis on mechanisms involving autoreactive T-cell recognition of HLA-B27 complexes displaying arthritogenic peptides, and more recently non-canonical recognition of abnormal forms of HLA-B27 free of beta(2)m (heavy-chain dimers or monomers), it remains unclear whether immunological recognition plays a role in pathogenesis. The recognition that the HLA-B27 heavy chain misfolds during assembly, and causes endoplasmic reticulum 'stress', has led to the observation that this activates the unfolded protein response. This has opened additional areas of investigation into the response of immune system cells to protein misfolding, and suggested novel alternative concepts that may explain the role of HLA-B27 in pathogenesis. This chapter will discuss available data and current concepts regarding the pathogenesis of ankylosing spondylitis.

The amino acid at position 97 is involved in folding and surface expression of HLA-B27.

HLA-B27 confers susceptibility to ankylosing spondylitis (AS) but the mechanism linking this association remains unknown. Other properties unrelated to its natural role of antigen presenting function may be important in disease pathogenesis. We determined here the impact of N97D substitution on the folding and expression of HLA-B*2704 transfected in the 721.221 cell line. The mutation at position 97 abolishes the surface expression of non-conformational (HC10) and conformational (ME1) forms. The expression of ME1 forms was found to be absent in B*2704 N97D by immunoprecipitation and flow cytometry of fixed and permeabilized cell experiments with the conformation-sensitive ME1 antibody. However, immunoblotting cell lysates with HC10 revealed the presence of unfolded heavy chain (HC) and HC-dimer forms. The impact of the N97D mutation in the exit from the endoplasmic reticulum (ER) was analysed by western blot after endoglycosidase-H treatment, and it was found that B*2704 N97D was retained and accumulated as unfolded molecules. We tested for mutant association with transporter associated with antigen processing (TAP), calnexin (CNX), calreticulin (CLR) and beta2 microglobulin (beta2m). The wild-type B*2704 and N97D mutants were associated with TAP, CNX and CLR, although HC10 forms of mutant N97D interact more weakly with TAP. Only folded molecules of HLA-B*2704 were associated with beta2m. Surprisingly, the peptide-binding assay demonstrated the ability of unfolded N97D molecules to bind high-affinity peptides. It has been suggested that AS may arise because of aberrant folding of HLA-B27 molecules within the ER. Future work must therefore aim to clarify the functional connection between the unfolded protein response pathway in response to the accumulation of HLA-B27 in the ER. This mutant could be useful as a model for the misfolding of HLA-B27.

Spondylarthritis-associated and non-spondylarthritis-associated B27 subtypes differ in their dependence upon tapasin for surface expression and their incorporation into the peptide loading complex.

OBJECTIVE: B27 subtypes associated with susceptibility to ankylosing spondylitis (AS), and those reported not to be associated with AS, are found to differ in the amino acids that are known in other HLA class I molecules to alter the requirements for tapasin and incorporation into the peptide loading complex. The purpose of this study was to examine the behavior of B*2704 and B*2705 in comparison with B*2706 and B*2709 during early events in HLA class I antigen expression, and determine if their behavior correlates with disease association. METHODS: Cell lines with nonfunctional tapasin were transiently transfected with different B27 subtypes and their site-directed mutants, and surface expression analyzed by flow cytometry. The association with the peptide loading complex was determined by immunoprecipitation of heterodimeric transporter-associated peptide and analysis of coprecipitated B27. RESULTS: Amino acids at positions 114, 116, and 152 in the different B27 subtypes were shown to perform key roles in defining a requirement for interaction with tapasin. Not all disease-associated alleles were expressed optimally in the absence of tapasin; furthermore, dependence on tapasin for cell surface expression did not correlate with disease association. Although B*2706, which is not associated with disease, exhibited a number of properties different from those of the disease-associated subtypes, these properties were not displayed by the non-disease-associated allele B*2709. CONCLUSION: These results indicate that the ability to exhibit optimal cell surface expression in the absence of tapasin is not a prerequisite for susceptibility to AS.