Dose HLA-B5, 7, 8, 27, and 51 Antigens Associated to Behcet's disease? A Study in Southwestern Iran.

BACKGROUND: Behcet's disease is a potentially life threatening autoimmune disease with recurrent ulcers and unknown pathogenesis. Gender and human leukocyte antigen-B51 seem to have an effective role in the clinical features of the disease. OBJECTIVE: The aim of this study is to evaluate the frequency of HLA-B5, 7, 8, 27 and 51 in behçet's disease in southwestern Iranian patients who visited the rheumatology clinic and to find the association between these HLA types and the disease. METHODS: 63 patients with behcet's disease participated in this study and peripheral blood samples were collected from them. The expression of each HLA antigen was evaluated by standard lymphocytotoxicity technique. RESULTS: Compared to other studied antigens, the expression of HLA-B5 and HLA-B51 was more prevalent among our patients. According to the results, 25% and 21% of patients were positive for HLA-B5 and HLA-B51, respectively. CONCLUSIONS: HLA-B5 and HLA-B51 are dominant positive HLA antigens among behcet's disease patients in the southwest of Iran; however, we cannot conclude that these antigens are valuable diagnostic or prognostic biomarkers due to our study limitations. We suggest studying the association between HLA-B antigens and inflammation severity in patients to determine the possible prognostic value of HLA-B antigens in Iranian population in the southwest and this region needs more studies in HLA subject among BD patients because of the frequency of BD to evaluate the value of HLA typing in BD prognosis.

Breadth and Dynamics of HLA-A2- and HLA-B7-Restricted CD8+ T Cell Responses against Nonstructural Viral Proteins in Acute Human Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8+ T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2- and HLA-B7-restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2- and HLA-B7-restricted Ag-specific CD8+ T cell response during the acute stages of human TBEV infection. HLA-A2- and HLA-B7-restricted TBEV epitope-specific effector cells predominantly displayed a CD45RA-CCR7-CD27+CD57- phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2- and HLA-B7-restricted TBEV-specific CD8+ T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2-restricted CD8+ T cell populations was CD45RA-CCR7-CD27+CD57+, whereas the HLA-B7-restricted CD8+ T cell population was predominantly CD45RA+CCR7-CD27+CD57+ Almost all TBEV epitope-specific CD8+ T cells expressed α4 and ß1 integrins at days 7 and 21, whereas the bulk CD8+ T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2- and HLA-B7-restricted TBEV epitope-specific CD8+ T cells.

Flow cytometric human leukocyte antigen-B27 typing with stored samples for batch testing.

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: WE COMPARED FOUR STORAGE METHODS: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Epidemiology of spondyloarthritis.

Spondyloarthritis (SpA) represents a group of interrelated diseases with common clinical features and a close association with HLA-B27. Reports of incidence and prevalence of diseases vary depending on methodological differences between studies, the case definition used to classify disease, and the prevalence of HLA-B27 in the population studied. Newly proposed criteria for axial SpA and peripheral SpA present a new approach to facilitate classification of the SpA into 2 main subtypes and the criteria allow earlier detection of patents with inflammatory back pain. These criteria were developed for use in a (specialized) clinical setting and not for large epidemiologic studies.

Teenage acute macular neuroretinopathy.

PURPOSE: To present the first case of acute macular neuroretinopathy in a teenager. METHODS: Case report, optical coherence tomography, fluorescein angiography, electroretinography. RESULTS: Optical coherence tomography, fluorescein angiography, and electroretinography were used to diagnose acute macular neuroretinopathy. Laboratory results were positive for HLA-B7. CONCLUSIONS: The rare diagnosis of acute macular neuroretinopathy should be considered in girls on oral contraceptives, with a recent history of acute viral illness, who present with acute rapid onset of multiple, paracentral scotomas.

Identification and epitope enhancement of a PAX-FKHR fusion protein breakpoint epitope in alveolar rhabdomyosarcoma cells created by a tumorigenic chromosomal translocation inducing CTL capable of lysing human tumors.

Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4-CD56-) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma.

Phenotypic diversity is not determined by independent genetic factors in familial spondylarthropathy.

OBJECTIVE: To analyze the segregation of manifestations belonging to the spectrum of spondylarthropathy (SpA) among patients and unaffected siblings within SpA multiplex families. METHODS: Ninety-five multiplex families have been investigated. The diagnosis of SpA was made according to European Spondylarthropathy Study Group criteria. The prevalence of SpA manifestations was determined in unaffected siblings and compared with their prevalence in patients. RESULTS: We compared 241 SpA patients with 259 unaffected siblings. The prevalence of skeletal and extraarticular features not used as diagnostic criteria, i.e., radiographic sacroiliitis, peripheral enthesitis, uveitis, psoriasis, and inflammatory bowel disease, was significantly increased in patients compared with unaffected siblings. This result was not accounted for by sex or HLA-B27 distribution differences. CONCLUSION: In familial SpA, skeletal and extraarticular manifestations tend to segregate together, implying that all subsets are predominantly determined by a shared component, and that accessory factors must be responsible for phenotype diversity.

[A case of Sjögren's syndrome complicated by polymyositis and sarcoidosis with HLA-B7 and DR 8: common causes of susceptibility for these diseases].

We describe a case of a Japanese patient initially presenting with Sjögren's syndrome who later developed polymyositis and sarcoidosis. A 67-year-old woman with a 4 month history of myalgia was admitted in April 1998 for examination. The patient had a 10 year history of symptoms consistent with Sjögren's syndrome. A diagnosis of polymyositis was made based on a biopsy of the muscle and an electromyogram. Positive Shirmer and Rose Bengal tests and results of a minor salivary gland biopsy were all consistent with Sjögren's syndrome. Chest computed tomography detected a bilateral hilar lymphadenopathy. Microscopic examination of a mediastinal lymph node demonstrated multiple noncaseating granulomas with multiple epithelioid cells and Langhans-like giant cells. A diagnosis of sarcoidosis was made based on these findings. Hepatitis C infection was also detected by elevated antibody levels. The patient was given 40 mg/day of oral prednisolone and a remission of her myositis and lymphadenopathy was obtained. The patient exhibited HLA-B7 and HLA-DR 8. HLA-DR 8 is commonly associated with these three disorders, and HLA-B7 is also associated with overlap syndrome in Japanese patients. The present case suggested the possibility of a common etiological background for these three disorders. Furthermore, the importance of genetic background, including HLA phenotype, in determining susceptibility to these disorders was demonstrated.

Donor-derived soluble HLA plasma levels can not be used to monitor graft rejection in heart transplant recipients.

OBJECTIVE: Increased levels of both donor- and recipient-derived HLA class I molecules (sHLA-I) can be found in serum or plasma of transplanted patients during rejection. Earlier data indicate that levels of donor-derived sHLA-I (dsHLA-I) correlate better with graft rejection than total sHLA Class I (Zavazava N, Kraatz E, Gassel AM, Muller-Ruchholtz W. Plasma MHC class I expression in cardiac graft patients: donor-specific soluble antigen in a pre-sensitized graft patient. Transplant Proc 1991;23:2258-2260; Claas FHJ, Jankowska-Gan E, DeVito LD, et al. Monitoring of heart transplant rejection using a donor-specific soluble HLA class I ELISA. Hum Immunol 1993;37:121). Therefore, quantifying donor-derived soluble counterparts of HLA Class I (sHLA-I) in the plasma of the recipient may offer a new possibility for non-invasive monitoring of rejection after organ transplantation. METHODS: In an extended study with 34 heart transplant recipients, we used sHLA-I specific ELISAs to monitor donor-derived soluble sHLA-A2, -A3, -A9, -B7, -B12 and B51. RESULTS: The assays were sensitive enough to detect dsHLA Class I in plasma of the recipients. However, the levels of sHLA were not found to be a useful tool for monitoring rejection. Rejection was often associated with low levels of donor sHLA. The recent finding that antibodies can inhibit the detection of sHLA molecules might explain this discrepancy. In order to test this hypothesis, patient sera were screened for the presence of anti-HLA antibodies and the results were related to the donor-derived sHLA levels. Only in four out of 34 patients HLA Class I specific antibodies could explain the low sHLA levels during rejection. CONCLUSIONS: In heart transplantation increased donor-derived sHLA levels are not a suitable marker for rejection and that antibody formation can not explain these results. Therefore, monitoring rejection episodes on the basis of donor-derived soluble HLA molecules is not a realistic approach to decrease the number of biopsies after heart transplantation.

Donor-derived human leukocyte antigen class I proteins in the serum of heart transplant recipients.

BACKGROUND: Human leukocyte antigen class I proteins are expressed on most cell types in all organ allografts but are constitutively secreted only by certain organs, for example, the liver. We hypothesized that detectable levels of donor-derived human leukocyte antigen proteins would be released from transplanted cardiac allografts only when the allograft was immunologically stimulated, that is, during rejection and perhaps during viral infection. If so, then the release of donor human leukocyte antigen might be a noninvasive monitor of these events. METHODS: We used an enzyme-linked immunosorbent assay to detect donor-derived human leukocyte antigen-A2 in the serum of 21 human leukocyte antigen-A2 negative recipients of human leukocyte antigen-A2-positive heart transplants. The level of donor human leukocyte antigen-A2 during the first 100 days after transplantation was correlated with the clinical status of the patient. RESULTS: We found little or no donor human leukocyte antigen in the serum of heart transplant recipients whose postoperative clinical course was unremarkable for infection or rejection. We did find donor-derived human leukocyte antigen in the serum of heart transplant recipients transiently in the week immediately after transplantation, continuously from patients in whom chronic rejection was developing, during cytomegalovirus infection, and during some, but not all, acute rejection episodes as determined by endomyocardial biopsy. CONCLUSIONS: These findings are consistent with the hypothesis that the donor human leukocyte antigen serum level reflects vascular diseases, rather than myocardial disease in the transplanted heart. Therefore, the serum level of donor human leukocyte antigen cannot be used as a monitor of cellular infiltration and myocyte damage as currently assessed by endomyocardial biopsy but may be an early indicator of the development of vascular disease such as chronic rejection.

Role of antibodies to tubulointerstitial nephritis antigen in human anti-tubular basement membrane nephritis associated with membranous nephropathy.

We report three patients, from two unrelated families, with anti-tubular basement membrane (TBM) antibody nephritis associated with membranous nephropathy. This rare disorder is characterized by nephrotic syndrome, tubular dysfunction, and progression to renal failure. Direct immunofluorescent studies in these patients revealed linear IgG deposition along the proximal TBM, while circulating antibodies reacting with proximal TBM but not with glomerular basement membrane were identified by indirect immunofluorescence. Sera from all three patients reacted by enzyme-linked immunosorbent assay and Western immunoblotting with purified 58-kd tubulointerstitial nephritis (TIN) antigen isolated from TBM. Additional reactivity with a 175-kd component, which may be a higher-molecular-weight form of TIN antigen, was observed by immunoblotting. Since recurrent Fanconi syndrome was seen after transplantation in one patient, anti-TBM antibodies were removed by plasmapheresis prior to kidney transplantation in the other two patients. Neither patient has clinical evidence of recurrent anti-TBM nephritis in the allograft despite the posttransplantation reappearance of anti-TBM antibodies in the serum of one patient. Serologic and molecular HLA class I and class II polymorphism analysis has identified the presence of both HLA-B7 and -DRw8 antigens in two unrelated affected individuals (0.3% expected frequency in the white population). We conclude that sera from patients with anti-TBM nephritis associated with membranous nephropathy react with 58-kd TIN antigen previously implicated in the pathogenesis of primary anti-TBM nephritis. This rare autoimmune disorder may be HLA associated with B7 and/or DRw8, providing susceptibility to the disease. Further investigation is needed to understand the pathogenesis of recurrent anti-TBM nephritis in the renal allograft.