Klinefelter's syndrome (47,XXY) is in excess among men with Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.

Mitochondrial Dysfunction and Fatigue in Sjögren's Disease.

Objectives: Sjögren's disease (SjD) is a common exocrine disorder typified by chronic inflammation and dryness, but also profound fatigue, suggesting a pathological basis in cellular bioenergetics. In healthy states, damaged or dysfunctional mitochondrial components are broken down and recycled by mitophagy, a specialized form of autophagy. In many autoimmune disorders, however, evidence suggests that dysfunctional mitophagy allows poorly functioning mitochondria to persist and contribute to a cellular milieu with elevated reactive oxygen species. We hypothesized that mitophagic processes are dysregulated in SjD and that dysfunctional mitochondria contribute to overall fatigue. We sought to link fatigue with mitochondrial dysfunction directly in SjD, heretofore unexamined, and further sought to assess the pathogenic extent and implications of dysregulated mitophagy in SjD. Methods: We isolated pan T cells via negative selection from the peripheral blood mononuclear cells of 17 SjD and 8 age-matched healthy subjects, all of whom completed fatigue questionnaires prior to phlebotomy. Isolated T cells were analyzed for mitochondrial oxygen consumption rate (OCR) and glycolysis using Seahorse, and linear correlations with fatigue measures were assessed. A mitophagy transcriptional signature in SjD was identified by reanalysis of whole-blood microarray data from 190 SjD and 32 healthy subjects. Differential expression analyses were performed by case/control and subgroup analyses comparing SjD patients by mitophagy transcriptional cluster against healthy subjects followed by bioinformatic interpretation using gene set enrichment analysis. Results: Basal OCR, ATP-linked respiration, maximal respiration, and reserve capacity were significantly lower in SjD compared to healthy subjects with no observed differences in non-mitochondrial respiration, basal glycolysis, or glycolytic stress. SjD lymphocytic mitochondria show structural alterations compared to healthy subjects. Fatigue scores related to pain/discomfort in SjD correlated with the altered OCR. Results from subgroup analyses by mitophagic SjD clusters revealed highly variable inter-cluster differentially expressed genes (DEGs) and expanded the number of SjD-associated gene targets by tenfold within the same dataset. Conclusion: Mitochondrial dysfunction, associated with fatigue, is a significant problem in SjD and warrants further investigation.

Dual effect of the macrophage migration inhibitory factor gene on the development and severity of human systemic lupus erythematosus.

OBJECTIVE: To study the effect of the innate cytokine macrophage migration inhibitory factor (MIF) on the susceptibility and severity of systemic lupus erythematosus (SLE) in a multinational population of 1,369 Caucasian and African American patients. METHODS: Two functional polymorphisms in the MIF gene, a -794 CATT(5-8) microsatellite repeat (rs5844572) and a -173 G/C single-nucleotide polymorphism (rs755622), were assessed for association with SLE in 3,195 patients and healthy controls. We also measured MIF plasma levels in relation to genotypes and clinical phenotypes, and assessed Toll-like receptor 7 (TLR-7)-stimulated MIF production in vitro. RESULTS: Both Caucasians and African Americans with the high-expression MIF haplotype -794 CATT(7)/-173*C had a lower incidence of SLE (in Caucasians, odds ratio [OR] 0.63, 95% confidence interval [95% CI] 0.53-0.89, P = 0.001; in African Americans, OR 0.46, 95% CI 0.23-0.95, P = 0.012). In contrast, among patients with established SLE, reduced frequencies of low-expression MIF genotypes (-794 CATT(5)) were observed in those with nephritis, those with serositis, and those with central nervous system (CNS) involvement when compared to patients without end-organ involvement (P = 0.023, P = 0.005, and P = 0.04, respectively). Plasma MIF levels and TLR-7-stimulated MIF production in vitro reflected the underlying MIF genotype of the studied groups. CONCLUSION: These findings suggest that MIF, which has both proinflammatory properties and macrophage and B cell survival functions, exerts a dual influence on the immunopathogenesis of SLE. High-expression MIF genotypes are associated with a reduced susceptibility to SLE and may contribute to an enhanced clearance of infectious pathogens. Once SLE develops, however, low-expression MIF genotypes may protect from ensuing inflammatory end-organ damage.

The lupus family registry and repository.

The Lupus Family Registry and Repository (LFRR) was established with the goal of assembling and distributing materials and data from families with one or more living members diagnosed with SLE, in order to address SLE genetics. In the present article, we describe the problems and solutions of the registry design and biometric data gathering; the protocols implemented to guarantee data quality and protection of participant privacy and consent; and the establishment of a local and international network of collaborators. At the same time, we illustrate how the LFRR has enabled progress in lupus genetics research, answering old scientific questions while laying out new challenges in the elucidation of the biologic mechanisms that underlie disease pathogenesis. Trained staff ascertain SLE cases, unaffected family members and population-based controls, proceeding in compliance with the relevant laws and standards; participant consent and privacy are central to the LFRR's effort. Data, DNA, serum, plasma, peripheral blood and transformed B-cell lines are collected and stored, and subject to strict quality control and safety measures. Coded data and materials derived from the registry are available for approved scientific users. The LFRR has contributed to the discovery of most of the 37 genetic associations now known to contribute to lupus through 104 publications. The LFRR contains 2618 lupus cases from 1954 pedigrees that are being studied by 76 approved users and their collaborators. The registry includes difficult to obtain populations, such as multiplex pedigrees, minority patients and affected males, and constitutes the largest collection of lupus pedigrees in the world. The LFRR is a useful resource for the discovery and characterization of genetic associations in SLE.

Klinefelter's syndrome (47,XXY) among men with systemic lupus erythematosus.

AIM: To determine the rate of Klinefelter's syndrome among men with systemic lupus erythematosus (SLE), and to determine whether the manifestations of SLE in these men are different from that seen in 46,XY men. METHODS: A total of 276 men with SLE underwent a real-time PCR procedure to screen for more than one X chromosome. Those with results consistent with two X chromosomes were further characterized by karyotype and FISH. Clinical manifestations of SLE were determined by interview, questionnaire and medical chart review. Each man with Klinefelter's and SLE was matched to four 46,XY men with SLE. Rates of SLE manifestations were compared with chi-square analyses. RESULTS: We found seven of the 286 men with SLE had Klinefelter's syndrome. Four of these seven were nonmosaic 47,XXY, while two were mosaic 46,XY/47,XXY and one was 46,XX/47,XXY. The men with 47,XXY did not have severe manifestations of SLE including no proliferative renal disease, neurological disease, thrombocytopenia, autoimmune haemolytic anaemia, discoid skin disease or anti-RNP/Sm. CONCLUSION: 47,XXY is found in excess among men with SLE. Men commonly have SLE that is more severe than that found among women, but the 47,XXY men had less severe SLE than other men.

Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome.

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Rare X Chromosome Abnormalities in Systemic Lupus Erythematosus and Sjögren's Syndrome.

OBJECTIVE: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. METHODS: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. RESULTS: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000-50,000 live female births, while partial triplications are even rarer. CONCLUSION: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative.

Significantly reduced lymphadenopathy, salivary gland infiltrates and proteinuria in MRL-lpr/lpr mice treated with ultrasoluble curcumin/turmeric: increased survival with curcumin treatment.

OBJECTIVES: Commercial curcumin (CU), derived from food spice turmeric (TU), has been widely studied as a potential therapeutic for a variety of oncological and inflammatory conditions. Lack of solubility/bioavailability has hindered curcumin's therapeutic efficacy in human diseases. We have solubilised curcumin in water applying heat/pressure, obtaining up to 35-fold increase in solubility (ultrasoluble curcumin (UsC)). We hypothesised that UsC or ultrasoluble turmeric (UsT) will ameliorate systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS)-like disease in MRL-lpr/lpr mice. METHODS: Eighteen female MRL-lpr/lpr (6 weeks old) and 18 female MRL-MpJ mice (6 weeks old) were used. Female MRL-lpr/lpr mice develop lupus-like disease at the 10th week and die at an average age of 17 weeks. MRL-MpJ mice develop lupus-like disease around 47 weeks and typically die at 73 weeks. Six mice of each strain received autoclaved water only (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group) or UsT (lpr-TU or MpJ-TU group) in the water bottle. RESULTS: UsC or UsT ameliorates SLE in the MRL-lpr/lpr mice by significantly reducing lymphoproliferation, proteinuria, lesions (tail) and autoantibodies. lpr-CU group had a 20% survival advantage over lpr-water group. However, lpr-TU group lived an average of 16 days shorter than lpr-water group due to complications unrelated to lupus-like illness. CU/TU treatment inhibited lymphadenopathy significantly compared with lpr-water group (p=0.03 and p=0.02, respectively) by induction of apoptosis. Average lymph node weights were 2606±1147, 742±331 and 385±68 mg, respectively, for lpr-water, lpr-CU and lpr-TU mice. Transferase dUTP nick end labelling assay showed that lymphocytes in lymph nodes of lpr-CU and lpr-TU mice underwent apoptosis. Significantly reduced cellular infiltration of the salivary glands in the lpr-TU group compared with the lpr-water group, and a trend towards reduced kidney damage was observed in the lpr-CU and lpr-TU groups. CONCLUSIONS: These studies show that UsC/UsT could prove useful as a therapeutic intervention in SLE/SS.

HLA alleles and haplotypes among the Lakota Sioux: report of the ASHI minority workshops, part III.

Human leukocyte antigen (HLA) class I and II alleles were defined for 302 Lakota Sioux American Indians as part of the American Society for Histocompatibility and Immunogenetics coordinated studies on minority populations. The study group was comprised of adult volunteers from the Cheyenne River and Ogala Sioux tribes residing, respectively, on the Cheyenne River and Pine Ridge Reservations in South Dakota. Of the participants, 263 (87%) claimed full American Indian ancestry through both maternal and paternal grandparents. The study group included 25 nuclear families that were informative for genotyping. HLA phenotypes from 202 adults with no other known first-degree relative included in the study were used for calculation of allele and haplotype frequencies by maximum likelihood estimation. HLA-A, -B, and -Cw alleles were found to be in Hardy Weinberg equilibrium. Deviation from equilibrium was observed for DRB1 alleles (p=0.01), but could be attributed to the sample size and the occurrence of some genotypes with low expected frequencies. Polymorphism among the Sioux was limited with four to seven alleles comprising >80% of those observed at each locus. Several alleles were found at high frequency (0.05-0.30) among the Sioux that are also prevalent in other Native Americans and Alaska Natives, including: A*2402, *3101, and *0206; B*3501,*3901, *5101, and *2705; Cw*0702, *0404, and *03041; DRB1*0407, *0404, *1402, and *16021; and DQB1*0301, *0302, and *0402. DRB1*0811, which has been only previously described in Navajo and Tlingit Indians, was found to occur at a frequency of 0.119 among the Sioux. Two new alleles were defined among the Sioux: Cw*0204 and DRB1*040703, which were found in two and four individuals, respectively. In the haplotype analyses, significant linkage disequilibrium (p<0.00001) was seen in all pairwise comparisons of loci and numerous two and three locus haplotypes were found to have strong, positive linkage disequilibrium values. The two most common extended haplotypes among the Sioux, determined by maximum likelihood estimation and genotyping were: A*31012, B*3501, Cw*0404, DRB1*0407; and A*24021, B*3501, Cw*0404, DRB1*0404.

Identification of unique microRNA signature associated with lupus nephritis.

MicroRNAs (miRNA) have emerged as an important new class of modulators of gene expression. In this study we investigated miRNA that are differentially expressed in lupus nephritis. Microarray technology was used to investigate differentially expressed miRNA in peripheral blood mononuclear cells (PBMCs) and Epstein-Barr Virus (EBV)-transformed cell lines obtained from lupus nephritis affected patients and unaffected controls. TaqMan-based stem-loop real-time polymerase chain reaction was used for validation. Microarray analysis of miRNA expressed in both African American (AA) and European American (EA) derived lupus nephritis samples revealed 29 and 50 differentially expressed miRNA, respectively, of 850 tested. There were 18 miRNA that were differentially expressed in both racial groups. When samples from both racial groups and different specimen types were considered, there were 5 primary miRNA that were differentially expressed. We have identified 5 miRNA; hsa-miR-371-5P, hsa-miR-423-5P, hsa-miR-638, hsa-miR-1224-3P and hsa-miR-663 that were differentially expressed in lupus nephritis across different racial groups and all specimen types tested. Hsa-miR-371-5P, hsa-miR-1224-3P and hsa-miR-423-5P, are reported here for the first time to be associated with lupus nephritis. Our work establishes EBV-transformed B cell lines as a useful model for the discovery of miRNA as biomarkers for SLE. Based on these findings, we postulate that these differentially expressed miRNA may be potential novel biomarkers for SLE as well as help elucidate pathogenic mechanisms of lupus nephritis. The investigation of miRNA profiles in SLE may lead to the discovery and development of novel methods to diagnosis, treat and prevent SLE.

Heterozygosity or homozygosity for 2 HLA class II haplotypes predict favorable outcomes for renal cell carcinoma treated with cytokine therapy.

PURPOSE: Durable responses to cytokine therapy occur in a small subset of patients with renal cell carcinoma. We determined if a common HLA genotype existed among these patients which might be associated with response and survival. MATERIALS AND METHODS: The study population consisted of 80 patients with metastatic renal cell carcinoma who had received cytokine therapy. DNA obtained from these patients was used for high resolution typing of HLA A, B, C, DRB1, DQA1 and DQB1 alleles. RESULTS: The class II alleles from patients with prolonged disease-free survival were predominantly composed of haplotype DRB1*0301/DQA1*0501/DQB1*0201 and DRB1*1501/DQA1*0102/DQB1*0602. The frequency of heterozygosity or homozygosity for these alleles was significantly greater in the good outcome group of patients than in those whose disease progressed during therapy. Heterozygosity or homozygosity at these loci was also associated with significant prolongation of survival. CONCLUSIONS: We conclude that heterozygosity or homozygosity for the class II haplotypes DRB1*0301/DQA1*0501/DQB1*0201 and DRB1*1501/DQA1*0102/DQB1*0602 is associated with durable response and survival in patients with metastatic renal cell carcinoma treated with cytokine therapy.