Spondyloarthropathies in eastern Asia.

The association between HLA-B27 and the spondyloarthropathies (SpAs) is so strong that it is supposed that the HLA-B27 molecule plays a pathogenetic role. In whites and Indonesians, the frequency of HLA-B27 is about 10%; in Chinese it is about 8%; and in Japanese it is only about 1%. The prevalence of SpA in the Chinese is at least 0.2%, but in native Indonesians, Philippinos, and Malaysians, SpA is rarely seen. Twenty-three subtypes (B*2701-B*2723) have been distinguished. These subtypes are not equally distributed over the world. In most countries the distribution of the subtypes among HLA-B27 SpA patients is the same as that among the normal HLA-B27-positive population. In China, the subtype B*2704 is frequent and the prevalence of SpA is high. Native Indonesians, however, mostly have subtype B*2706, and SpA is rarely seen in this population. It was shown that B*2706, probably like B*2709 in Sardinia, is not associated with SpA. The difference between the SpA-associated and non-SpA-associated subtypes is limited to only two amino acid residues (114 and 116) at the bottom of the peptide-binding groove of HLA-B27. This small difference between health and disease rewards studies for different peptide-binding capacities and may help us characterize the peptides that are involved in the pathogenesis of SpA. The differences in disease associations in these countries also have clinical implications. In Southeast Asia, HLA-B27 typing without subtyping has less clinical usefulness than in parts of the world where B*2706 is rarely seen. When native Indonesians, Malaysians, or Philippinos are suspected of having ankylosing spondylitis or a related SpA, it is worth asking if they had white or Chinese ancestors. If native HLA-B27-positive Indonesians (with subtypes other than B*2706) develop SpA, the clinical features are not different from those in other parts of the world. In the Chinese population on the mainland and in Taiwan, juvenile SpA is frequently seen. The onset is often a peripheral arthritis or enthesitis.

Evidence that HLA-B*2706 is not protective against spondyloarthropathy.

OBJECTIVE: Studies in Southeast Asia showed that HLA-B*2704 is positively associated with spondyloarthropathy (SpA), while B*2706 does not occur in such patients. In view of the absence of an association between B*2706 and SpA it was suggested that B*2706 protects against the disease, while it is supposed that B*2704 presents pathogenetic peptides. We studied families in which both B*2704 and B*2706 occurred to see whether in B*2704/B*2706 heterozygotes the effect of one of the subtypes shows a preponderance over the other. METHODS: Two families of mixed Chinese/Indonesian origin were studied. HLA-B27 subtyping was performed by polymerase chain reaction in combination with sequence specific oligonucleotide probes. RESULTS: In one family, members with B*2704, B*2706, or both occurred. In the other family B*2704, B*2706, and B*2708 were present. In both families SpA was seen only in B*2704 positive members, while the B*2706 and B*2708 positive members were healthy, except some B*2704/B*2706 or B*2704/B2708 heterozygotes. CONCLUSION: The pathogenic influence of B*2704 is thus dominant over the supposed protective influence of B*2706. It is probable that B*2704 can present pathogenetic peptides, while a protective influence of B*2706 does not exist. B*2708, which was until now described in only a few cases, behaved in this study as B*2706 and is probably not associated with SpA.

Clinical features of spondyloarthropathy in Chinese and native Indonesians.

The prevalence of spondyloarthropathy (SpA) in Chinese Indonesians is much higher than in native Indonesians. This is due to HLA-B27 subtype differences. In the present study we re-examined the clinical features of SpA in Indonesians to see whether, besides the HLA-B27 subtype differences, other factors affect the frequency of SpA. Seventy two patients with SpA were re-examined. The patients came from two clinics for rheumatic diseases. The overall entry ratio of Chinese to native Indonesians was 1:2. Ankylosing spondylitis (AS) was more frequent among the Chinese (n = 32, 94% B27 positive) than among the native Indonesians (n = 5, 40% B27 positive). HLA-B27 subtyping was performed on 22 of the 37 HLA-B27-positive AS patients. Twenty Chinese were positive for B*2704 and two native Indonesians were B*2705 positive. The clinical features of AS and reactive arthritis (ReA) showed no differences between the two populations and were similar to the clinical descriptions in other parts of the world. In conclusion, it can be stated that in spite of HLA-B27 subtype differences the clinical features of SpA in Chinese and native Indonesians are fully comparable.

Acute anterior uveitis and spondyloarthropathies.

Acute anterior uveitis (AAU) is characterized by sudden-onset, mostly unilateral exacerbations of an inflammation of the iris and ciliary body. The duration of illness is short if the patient is treated with corticosteroids. Half of all patients with any type of anterior uveitis are HLA-B27-positive, and more than half of the B27-positive patients have spondyloarthropathy. Ophthalmologists should therefore refer all patients with AAU who are HLA-B27-positive to a rheumatologist. Because attacks of AAU are extremely painful and frightening, most spondyloarthropathy patients with AAU will seek out an ophthalmologist on their own. The anterior chamber of the eye and the joints are mesenchymal cavities that are cleaned by macrophages. Anterior chamber-associated immune deviation is the mechanism by which specific regulatory T cells normally produce sufficient transforming growth factor-beta to impair inflammatory reactions that might hamper vision. Another mechanism of immune privilege is Fas-ligand induced apoptosis. Because the cells of the anterior eye express Fas-ligand, infiltrating cells are apoptotically killed. Comparable mechanisms may occur at a lower level in joints. The cause of AAU and spondyloarthropathy is unknown. B27 is probably only responsible for one quarter of the pathogenesis, other non-B27 genetic factors for another quarter, and unknown exogenous factors for the remaining half. It is possible that Gram-negative bacteria such as Klebsiella or Yersinia are involved in the pathogenesis in a yet unknown way.

Antibodies to Klebsiella pneumoniae in Dutch patients with ankylosing spondylitis and acute anterior uveitis and to Proteus mirabilis in rheumatoid arthritis.

OBJECTIVE: To determine whether the association between increased humoral reactivity against Klebsiella and HLA-B27 associated diseases could be confirmed in Dutch patients with ankylosing spondylitis (AS) and acute anterior uveitis (AAU). METHODS: Under coded conditions sera from Dutch patients with AS, AAU, and rheumatoid arthritis (RA) and from HLA-B27 positive and negative healthy controls were studied for IgA anti-Klebsiella (K54) and IgG anti-Proteus antibodies with the indirect immunofluorescence assay on whole bacteria fixed in suspension with paraformaldehyde. Each group consisted of at least 17 sera. RESULTS: IgA anti-Klebsiella antibody titers were elevated in AS and HLA-B27 negative AAU compared to the HLA-B27 positive and negative controls or patients with active RA (p < 0.001). Furthermore, patients with active RA had elevated levels of IgG antibodies against P. mirabilis compared to every other test or control group (p < 0.001). There was no significant difference between the AS and RA patients in terms of serum C-reactive protein levels, although these were significantly elevated in both compared to healthy controls (p < 0.001), suggesting that the antibody elevations were not due to a nonspecific inflammatory effect. The same sera were blindly tested with negative results by 2 other centers. The discrepancies are probably the result of differences in the methods used. CONCLUSION: Our data support the hypothesis that Klebsiella are involved in the pathogenesis of AS and AAU and that the same might be true for Proteus in RA.

Range of antinuclear antibodies in "healthy" individuals.

OBJECTIVE: To determine the range of antinuclear antibodies (ANA) in "healthy" individuals compared with that in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjögren's syndrome (SS), rheumatoid arthritis (RA), or soft tissue rheumatism (STR). METHODS: Fifteen international laboratories experienced in performing tests for ANA by indirect immunofluorescence participated in analyzing coded sera from healthy individuals and from patients in the 5 different disease groups described above. Except for the stipulation that HEp-2 cells should be used as substrate, each laboratory used its own in-house methodology so that the data might be expected to reflect the output of a cross-section of worldwide ANA reference laboratories. The sera were analyzed at 4 dilutions: 1:40, 1:80, 1:160, and 1:320. RESULTS: In healthy individuals, the frequency of ANA did not differ significantly across the 4 age subgroups spanning 20-60 years of age. This putatively normal population was ANA positive in 31.7% of individuals at 1:40 serum dilution, 13.3% at 1:80, 5.0% at 1:160, and 3.3% at 1:320. In comparison with the findings among the disease groups, a low cutoff point at 1:40 serum dilution (high sensitivity, low specificity) could have diagnostic value, since it would classify virtually all patients with SLE, SSc, or SS as ANA positive. Conversely, a high positive cutoff at 1:160 serum dilution (high specificity, low sensitivity) would be useful to confirm the presence of disease in only a portion of cases, but would be likely to exclude 95% of normal individuals. CONCLUSION: It is recommended that laboratories performing immunofluorescent ANA tests should report results at both the 1:40 and 1:160 dilutions, and should supply information on the percentage of normal individuals who are positive at these dilutions. A low-titer ANA is not necessarily insignificant and might depend on at least 4 specific factors. ANA assays can be a useful discriminant in recognizing certain disease conditions, but can create misunderstanding when the limitations are not fully appreciated.

HLA-B27 subtypes positively and negatively associated with spondyloarthropathy.

OBJECTIVE: Eleven subtypes of HLA-B27 have been identified. If some of these subtypes had a stronger association with spondyloarthropathy (SpA) than others, this might tell us which peptides are of pathogenetic importance. A subtype preponderance has not been proved in Caucasians or in Asian Indians. Our objective was to determine whether some subtypes are positively or negatively associated with SpA in Indonesia. METHODS: Cells of 34 HLA-B27 positive patients with SpA (fulfilling the European Spondylarthropathy Study Group criteria) and 26 HLA-B27 positive controls, all living in Java, Indonesia, were sampled. Patients and controls were divided according to their presumed ethnic origin. HLA-B27 subtyping (B*2701-09) was performed by polymerase chain reaction in combination with sequence specific oligonucleotide probes to analyze polymorphism in exons 2 and 3 of HLA-B27. RESULTS: HLA-B*2701, *2702, *2703, *2708, and *2709 were found in neither group. HLA-B*2704 was found in 23/34 (68%) of the patients and in only 4/26 (15%) of the controls (p < 0.01). HLA-B*2706 was found in none of the 34 patients, but in 21/26 (81%) of the controls (p < 0.01). One drawback of the study was that most patients were of Chinese descent and most controls were native Javanese. Nevertheless, the absence of SpA among HLA-B*2706 positive individuals is noteworthy. CONCLUSION: HLA-B*2704 is positively associated with SpA (RR = 11.5), while *2706 is negatively associated with this disease (RR < 0.007). The results confirm the findings of Lopez-Larrea, et al in Thailand.

Long-term course of tear gland function in patients with keratoconjunctivitis sicca and Sjögren's syndrome.

AIMS: To assess the course of tear gland function of patients with keratoconjunctivitis sicca (KCS) associated with primary (KCS-PSS) or secondary Sjögren's syndrome (KCS-SSS), and of patients with KCS not related to Sjögren's syndrome (KCS-NS). METHODS: In 106 patients with dry eye an ophthalmic diagnosis of KCS was made. Subsequent evaluations revealed a diagnosis of KCS-PSS in 31, KCS-SS in 19, and KCS-NS in 56 patients. Follow up assessments have been performed 10-12 years after initial diagnosis. RESULTS: At baseline and at follow up tear gland function tests were worse in patients with KCS-PSS compared with the other forms of KCS. At follow up in the KCS-SSS patient group the tear gland function variables returned to marginal normal limits. In contrast with expectation, a marked improvement of the tear gland function variables in the KCS-NS patient group was noted. CONCLUSIONS: In KCS-PSS patients tear gland function is characterised by a steady state situation. In KCS-SSS patients the normalisation of tear gland function variables most probably reflects a remission of the underlying disease. In view of the overall improvement in KCS-NS patients the term age related KCS should be avoided.

Antinuclear antibody determination in a routine laboratory.

Pitfalls in the method for demonstrating antinuclear antibodies (ANA) by the indirect immunofluorescence technique are described and the use of international standard preparations outlined. Determination of the optimal border dilution dividing positive from negative results is discussed. Each laboratory is a unique setting; it must define its own method, which should rarely be changed. One should not rely on copying methods from other laboratories or commercial firms, but the reproducibility of the nuclear substrate, the conjugate, and other variables should be controlled daily by the use of a control serum which has been related to the WHO standard preparation for ANA of the homogeneous type. Since many sera contain mixtures of different ANA, the results of routine tests are best expressed in titres or expressions of the intensity of fluorescence. The ANA test using the immunofluorescence technique should be used as a screening method for other tests allowing a more defined interpretation of the ANA. Each laboratory should individually determine the border between positive and negative results. Therefore about 200 sera from local healthy controls equally distributed over sex and age, and 100 sera from local patients with definite SLE should be tested. Since the local clinicians should become acquainted with this border it should rarely be changed. Finally each laboratory should participate regularly in national and international quality control rounds, where sera known to be difficult to interpret are tested. The judgment of the organisers of these rounds should stimulate improvements in the participating laboratories.

Long-term followup of patients with Sjögren's syndrome.

OBJECTIVE: To assess long-term outcome in patients with isolated keratoconjunctivitis sicca (KCS), primary Sjögren's syndrome (SS), and secondary SS. METHODS: In 112 patients referred because of dry eyes, an ophthalmologic diagnosis of KCS was made based on results of the Schirmer I test, the tear fluid lysozyme concentration, and rose bengal staining. Subsequent assessments, including sublabial salivary gland biopsy, were performed. Followup assessments were performed 10-12 years after initial diagnosis. RESULTS: Six patients were excluded because no biopsy specimen was available. Seventy-three percent of the remaining 106 patients were female, with a mean age of 53.5 years and a mean symptom duration of 3.9 years. Application of the 1987 classification criteria of Daniels and Talal revealed a diagnosis of isolated KCS in 56 patients, primary SS in 31, and secondary SS in 19. At baseline, 2 of 56 patients with isolated KCS and 8 of 31 with primary SS exhibited mild features of organ-specific autoimmune disease. At followup, 2 of 38 patients with isolated KCS and 4 of 21 with primary SS had developed new features related to autoimmune disease, not necessitating treatment with corticosteroids; none of the patients developed major glandular complications. Three of 30 patients with primary SS died of malignant lymphoma. In 1 of these patients, the possibility could not be excluded that sicca symptoms and infiltrates seen on sublabial salivary gland biopsy had occurred concomitantly with early stages of lymphoma. Malignant lymphoma did not develop in any of the patients with isolated KCS or secondary SS. CONCLUSION: Primary Sjögren's syndrome is characterized by a stable and rather mild course of glandular and extraglandular manifestations, in marked contrast to the increased risk of development of malignant lymphoma in these patients. Since patients with isolated KCS do not have an increased risk for development of malignant lymphoma, a presumptive diagnosis of primary SS should be confirmed in patients with sicca syndrome.

Seronegative spondyloarthropathies and HLA-B27 subtyes: a study in Asian Indians.

In this study, 60 HLA-B27+ve SSA patients and 17 healthy controls belonging to North India were analyzed to ascertain heterogeneity of the B27 molecule in this population. ID-IEF and PCR-SSOP technologies were used to analyze polymorphism in exon 2 and 3 of the HLA-B27 gene. Four different subtypes were encountered: B*2702,04,05 and 07. Other subtypes of B27 viz B*2701,03,06 and 08 were not encountered. B*2704 (common oriental subtype) and B*2705 (common Caucasian subtype) were the most common subtypes in the control and patient groups. B*2707 was less frequently encountered in both groups and B*2702 was found in only one AAU patient. B*2704 was the predominant subtype in the AS group (70.8%) compared to its frequency of 47% in healthy controls (RR = 2.73) while in the undiff SpA group, B*2705 occurred most frequently (73.1%, RR = 3.05). B27 subtypes segregated differently in males and females. 12 of the 17 male AS patients carried B*2704 as compared to 1 of 8 healthy males (X2 = 3.9, P < 0.05). On the other hand, in the undiff SpA, B*2705 was significantly raised in female patients (100%) as compared to healthy females (22.2%, X2 = 4.9, P < 0.05). Subtype distribution is indicative of racial admixture in the Asian Indian population.

Non-HLA-B27 genetic factors in HLA-B27 associated diseases.

Family and twin studies showed that besides HLA-B27 other genetic factors are of significance for the pathogenesis of ankylosing spondylitis, reactive arthritis or acute anterior uveitis. There are up to now no strong indications that any subtype of B27 is especially associated with these diseases. Also the indications that B2703 and B2706 are protective should be further substantiated. HLA-B27/HLA-B60 heterozygotes have a three times higher risk to develop ankylosing spondylitis as persons with another B type opposite B27. Haplotype studies made it unlikely that the other genetic factors are situated on chromosome 6p. Studies on 23 other genetic marker systems were all negative in this respect.

IgA antibodies against Klebsiella and other Gram-negative bacteria in ankylosing spondylitis and acute anterior uveitis.

Mucosal infections, especially of the gastrointestinal tract, are thought to trigger the onset and/or reactivation of ankylosing spondylitis (AS). Previous investigations into the role of Klebsiella and other Gram-negative bacteria in AS patients show contrasting results. In the present study prevalence of IgA antibodies against Klebsiella, Yersinia, Salmonella, Shigella, and Campylobacter was examined in serum samples from 30 patients having HLA-B27 associated ankylosing spondylitis, 32 patients with HLA-B27 associated acute anterior uveitis (AAU), and 27 HLA-B27 positive patients having both AS and AAU. Numbers of antibodies were compared with those in sera from 29 HLA-B27 negative patients with AAU, 26 healthy HLA-B27 positive and 31 HLA-B27 negative controls. IgA antibodies were detected using an indirect immunofluorescence assay on whole bacteria. In case of Yersinia, Salmonella, Shigella and Campylobacter, reference strains were used. Examination for anti-Klebsiella antibodies was performed using three different strains, isolated from patients with ankylosing spondylitis. The sera were tested on antibodies against Klebsiella K43 (BTS1) as well. The number of IgA positive sera against Yersinia, Salmonella, Shigella, Campylobacter and Klebsiella K43 (BTS1) did not differ between HLA-B27 positive patients and controls, nor among the various groups. Differences were neither observed when the Klebsiella strains from AS patients had been used as antigen. These results do not confirm a relationship between HLA-B27 associated AS or AAU and infection with Klebsiella or other Gram-negative bacteria.

IgA antibodies in HLA-B27 associated acute anterior uveitis and ankylosing spondylitis.

Acute anterior uveitis (AAU) and ankylosing spondylitis (AS) are, like reactive arthritis (ReA), strongly associated with HLA-B27. Mucosal infections play a role in the pathogenesis of ReA. To investigate whether these microorganisms are also involved in the pathogenesis of AAU and AS, we examined blood samples from patients with AAU, AS or both, and healthy controls for presence of antibodies against Klebsiella pneumoniae (K 30), Salmonella enteritidis and S. typhimurium, Chlamydia trachomatis, Proteus mirabilis and Borrelia burgdorferi. The IgA, IgG and IgM classes of these antibodies were measured using an enzyme-linked immunosorbent assay. No significant differences were found in the frequency in which these antibodies occurred in HLA-B27 positive patients with AAU or AS and healthy controls. However, IgA antibodies against K. pneumoniae (p < 0.01) and IgA and IgG antibodies against P. mirabilis (p < 0.01 and p < 0.05) were detected more frequently in HLA-B27 negative patients with AAU than in healthy controls. The results of this study are in contrast with various earlier reports in which antibodies against Klebsiella strains were more frequently found in patients with HLA-B27 associated ankylosing spondylitis than in healthy controls.

Comparison of peptides eluted from the groove of HLA-B27 from Salmonella infected and non-infected cells.

Reactive arthritis (ReA) is associated with the MHC class-I molecule HLA-B27 and caused by certain Gram-negative bacteria. The mechanism by which HLA-B27 confers a higher susceptibility for this disease compared to other MHC Class-I alleles is still not known. We investigated whether infection of human HLA-B27+ cells is able to change the peptide repertoire presented by these HLA-B27 molecules. To this end large quantities of a B-cell line (C1R-B27) transfected with HLA-B2705 were infected with S. typhimurium. Peptides were eluted from the B27 molecules and separated by Reversed Phase Chromatography (RPC). We then compared the peptide profiles obtained from S. typhimurium infected CIR B-cells with that obtained from non infected cells. Apart from a few additional peaks present in the profile derived from the infected batch the peptide profiles were almost identical. A few fractions were subjected to sequencing by Edman degradation. All peptides found were nonameres with arginine (Arg) at position 2 which is in agreement with the previously described HLA-B27 peptide binding motif. The majority of peaks expressed a mixture of at least four different peptides. The analysis of differences between HLA-B27 bound peptides from Salmonella infected and non infected cells might lead to the identification of T-cell epitopes shared by Salmonella and autoantigens.

Heterogeneity of rearranged T cell receptor V alpha and V beta gene transcripts in synovial fluid T cells of HLA-B27 positive reactive arthritis patients.

To analyze the T cell receptor (TCR) V-alpha/beta gene usage by synovial fluid (SF) and peripheral blood (PB) T cells of HLA-B27+ reactive arthritis (ReA) patients. The TCR V-alpha/beta gene usage was determined by the polymerase chain reaction on freshly isolated SF and PB mononuclear cells (MNC) of five HLA-B27+ ReA patients. A total of 30 TCR V alpha and 23 V beta (sub)family specific primers in combination with a C alpha or C beta specific primer, respectively, were used. In five patients most of the TCR V alpha and V beta gene segments expressed by PB T cells were also detected in the paired SF samples. Although one patient showed an increased expression of TCR V alpha2 in SF when compared to PB, the SF samples showed a heterogeneous TCR V-gene repertoire similar to PB. Although this study was limited to a small group of patients, the apparent lack of a restricted TCR V-gene repertoire in SF does not support the involvement of a single or limited number of T cell subsets in the disease process of HLA-B27+ ReA patients.

Spondylarthropathy and selective IgA deficiency.

No serum IgA was detected in a young male patient suffering from spondylarthropathy (SpA) with bilateral sacroiliitis arthritis, enthesopathy and inflammatory low back pain, whose symptoms occurred in reaction to a sexually induced urethritis. After a period of several months in which the spondylarthropathy was active, disease activity came to a rest. Three years later no progression of the SpA was observed. This finding might be an indication that IgA is not involved in the pathogenesis of spondylarthropathy.