Passive transfer of antibodies to the linear epitope 60 kD Ro 273-289 induces features of Sjögren's syndrome in naive mice.

Sjögren's syndrome (SS) is an autoimmune inflammatory disease that primarily affects the lacrimal and salivary glands causing dry eyes and mouth. Antibodies to Ro60 are observed frequently in patients with SS; however, the role of these antibodies in SS initiation and progression remains unclear. The sequence Ro60 273-289 (Ro274) is a known B cell epitope of Ro60 and antibodies to this epitope have been observed in a subset of SS patients and in animals immunized with Ro60 protein. Animals immunized with Ro274 linear peptide develop a Sjögren's-like illness. We hypothesized that passive transfer of anti-Ro274-specific immunoglobulin (Ig)G would induce a Sjögren's-like phenotype. To evaluate this hypothesis, we adoptively transferred affinity-purified Ro274 antibodies into naive BALB/c animals, then evaluated salivary gland histology, function and IgG localization 4 days post-transfer. At this time-point, there was no demonstrable mononuclear cell infiltration and salivary glands were histologically normal, but we observed a functional deficit in stimulated salivary flow of animals receiving Ro274 antibodies compared to animals receiving control IgG. Cellular fractionation and enzyme-linked immunosorbent assay revealed Ro274-specific antibodies in the nucleus and cytoplasmic fractions of isolated parotid salivary gland cells that was confirmed by immunohistochemistry. These data support the hypothesis that antibodies to Ro274 deposit in salivary glands can enter intact salivary gland cells and are involved in the dysregulation of salivary flow in SS.

Immunization with 60 kD Ro peptide produces different stages of preclinical autoimmunity in a Sjögren's syndrome model among multiple strains of inbred mice.

Sjögren's syndrome is a chronic illness manifested characteristically by immune injury to the salivary and lacrimal glands, resulting in dry mouth/eyes. Anti-Ro [Sjögren's syndrome antigen A (SSA)] and anti-La [Sjögren's syndrome antigen B (SSB)] autoantibodies are found frequently in Sjögren's subjects as well as in individuals who will go on to develop the disease. Immunization of BALB/c mice with Ro60 peptides results in epitope spreading with anti-Ro and anti-La along with lymphocyte infiltration of salivary glands similar to human Sjögren's. In addition, these animals have poor salivary function/low saliva volume. In this study, we examined whether Ro-peptide immunization produces a Sjögren's-like illness in other strains of mice. BALB/c, DBA-2, PL/J, SJL/J and C57BL/6 mice were immunized with Ro60 peptide-274. Sera from these mice were studied by immunoblot and enzyme-linked immunosorbent assay for autoantibodies. Timed salivary flow was determined after pharmacological stimulation, and salivary glands were examined pathologically. We found that SJL/J mice had no immune response to the peptide from Ro60, while C57BL/6 mice produced antibodies that bound the peptide but had no epitope spreading. PL/J mice had epitope spreading to other structures of Ro60 as well as to La, but like C57BL/6 and SJL/J had no salivary gland lymphocytic infiltration and no decrement of salivary function. DBA-2 and BALB/c mice had infiltration but only BALB/c had decreased salivary function. The immunological processes leading to a Sjögren's-like illness after Ro-peptide immunization were interrupted in a stepwise fashion in these differing mice strains. These data suggest that this is a model of preclinical disease with genetic control for epitope spreading, lymphocytic infiltration and glandular dysfunction.

Role of MYH9 and APOL1 in African and non-African populations with lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected P-value of P < 2.03 × 10(-3) were observed between LN and MYH9 in EAs (N = 4620), with the most pronounced association at rs2157257 (P = 4.7 × 10(-4), odds ratio (OR) = 1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, P = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population, and presents novel insight into the potential role of MYH9 in LN in EAs.

A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni-corrected threshold (P<1 × 10(-4)). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P=0.0004) and UBCH7 protein expression (P=0.0068). The results suggest that variants carried on the SLE-associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.

Identification of novel coding mutation in C1qA gene in an African-American pedigree with lupus and C1q deficiency.

OBJECTIVES: Homozygous C1q deficiency is an extremely rare condition and strongly associated with systemic lupus erythematosus. To assess and characterize C1q deficiency in an African-American lupus pedigree, C1q genomic region was evaluated in the lupus cases and family members. METHODS: Genomic DNA from patient was obtained and C1q A, B and C gene cluster was sequenced using next generation sequencing method. The identified mutation was further confirmed by direct Sanger sequencing method in the patient and all blood relatives. C1q levels in serum were measured using sandwich ELISA method. RESULTS: In an African-American patient with lupus and C1q deficiency, we identified and confirmed a novel homozygote start codon mutation in C1qA gene that changes amino acid methionine to arginine at position 1. The Met1Arg mutation prevents protein translation (Met1Arg). Mutation analyses of the patient's family members also revealed the Met1Arg homozygote mutation in her deceased brother who also had lupus with absence of total complement activity consistent with a recessive pattern of inheritance. CONCLUSION: The identification of new mutation in C1qA gene that disrupts the start codon (ATG to AGG (Met1Arg)) has not been reported previously and it expands the knowledge and importance of the C1q gene in the pathogenesis of lupus especially in the high-risk African-American population.

Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors have a role. Rare mutations in the TREX1 gene, the major mammalian 3'-5' exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurological condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls. A total of 40 single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene, were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied, and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P-values, false-discovery rate q values, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient, whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (minor allele frequency (MAF)>10%) revealed a relatively common risk haplotype in European SLE patients with neurological manifestations, especially seizures, with a frequency of 58% in lupus cases compared with 45% in normal controls (P=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (P=2.99E-13, OR=5.2, 95% CI=3.18-8.56). Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.

Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity.

Autoantibodies from many patients with systemic lupus erythematosus bind the Sm autoantigen B/B' polypeptide. The binding of serial serum specimens to the 233 overlapping octapeptides of Sm B/B' have shown that of the B/B'-derived octapeptides, PPPGMRPP and PPPGIRGP are early targets of the autoimmune response in some lupus patients. Rabbits immunized with PPPGMRPP and PPPGIRGP develop antibodies which not only bind these octapeptides, but also subsequently bind many other octapeptides of Sm B/B'. Eventually, the rabbits immunized with one octapeptide develop autoantibodies that bind other spliceosomal proteins including D, 70K, A, and C. Any mechanisms that operate to maintain tolerance or anergy for the spliceosome are thus overcome. Features considered typical of human systemic lupus erythematosus are also found in these peptide-immunized animals, such as antinuclear antibodies, anti-Sm precipitins, anti-double-stranded DNA, thrombocytopenia, seizures, and proteinuria. This disease model provides access to a mechanism for the development of humoral autoimmunity and may provide a basis to explain the immunopathogenesis of lupus in humans.

Review: Male systemic lupus erythematosus: a review of sex disparities in this disease.

Although males with systemic lupus erythematosus (SLE) represent 4-22% of all SLE patients, it may not be appropriate that these cases should be subordinated to females with SLE in terms of most health-related issues. Over the past few decades, some distinctive features of male lupus have been observed with regard to genetic and environmental aspects of sex differences, clinical features, and outcome. In addition, recent insights into sex disparities in this disease have brought forth a few plausible and novel pathogenetic hypotheses. This review discusses these findings and sex disparities in SLE that appear to be especially noteworthy and pertinent to our understanding of male SLE.

Complete complement deficiency in a large cohort of familial systemic lupus erythematosus.

Genetic complete deficiency of the early complement components such as C1, C2 and C4 commonly results in a monogenetic form of systemic lupus erythematosus (SLE). However, previous studies have examined groups of complete complement deficient subjects for SLE, while a familial SLE cohort has not been studied for deficiencies of complement. Thus, we undertook the present study to determine the frequency of hereditary complete complement deficiencies among families with two or more SLE patients. All SLE patients from 544 such families had CH50 determined. Medical records were examined for past CH50 values. There were 66 individuals in whom all available CH50 values were zero. All but four of these had a SLE-affected relative with a non-zero CH50; thus, these families did not have monogenetic complement deficient related SLE. The four remaining SLE-affected subjects were in fact two sets of siblings in which three of the four SLE patients had onset of disease at <18 years of age. Both patients in one of these families had been determined to have C4 deficiency, while the other family had no clinical diagnosis of complement deficiency. In this second family, one of the SLE patients had had normal C4 and C3 values, indicating that either C1q or C2 deficiency was possible. Thus, only 2 of 544 SLE families had definite or possible complement deficiency; however, 1 of 7 families in which all SLE patients had pediatric onset and 2 of 85 families with at least 1 pediatric-onset SLE patent had complete complement deficiency. SLE is found commonly among families with hereditary complement deficiency but the reverse is not true. Complete complement deficiency is rare among families with two or more SLE patients, but is concentrated among families with onset of SLE prior to age 18.

46,X,del(X)(q13) Turner's syndrome women with systemic lupus erythematosus in a pedigree multiplex for SLE.

Systemic lupus erythematosus (SLE) disproportionately affects women. Recent work demonstrates that men with Klinefelter's syndrome (47,XXY men) have a similar risk of developing SLE as do women. We present an unusual African-American family with two SLE-affected individuals in which one of the patients with SLE also has Turner's syndrome (46,X,del(X)(q13)). Although not definitive, this family raises interesting questions regarding the function of genes located on the X chromosome in the development of SLE. The paucity of case reports documenting the overlap of SLE with Turner's syndrome while there is an association of male SLE with Klinefelter's syndrome suggests a lower risk of SLE in women with Turner's syndrome. These observations are consistent with a gene dose effect at X with two X chromosomes (46,XX or 47,XXY) conferring higher risk and one X chromosome (46,XY or 45,XO) conferring lower risk of SLE.

Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-derived population.

Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T-cell, B-cell and accessory cell functions. B cells are hyperactive in SLE patients. An adapter protein expressed in B cells called BANK1 (B-cell scaffold protein with ankyrin repeats) was reported in a previous study to be associated with SLE in a European population. The objective of this study was to assess the BANK1 genotype-phenotype association in an independent replication sample. We genotyped 38 single nucleotide polymorphisms (SNPs) in BANK1 on 1892 European-derived SLE patients and 2652 European-derived controls. The strongest associations with SLE and BANK1 were at rs17266594 (corrected P-value=1.97 x 10(-5), odds ratio (OR)=1.22, 95% CI 1.12-1.34) and rs10516487 (corrected P-value=2.59 x 10(-5), OR=1.22, 95% CI 1.11-1.34). Our findings suggest that the association is explained by these two SNPs, confirming previous reports that these polymorphisms contribute to the risk of developing lupus. Analysis of patient subsets enriched for hematological, immunological and renal ACR criteria or the levels of autoantibodies, such as anti-RNP A and anti-SmRNP, uncovers additional BANK1 associations. Our results suggest that BANK1 polymorphisms alter immune system development and function to increase the risk for developing lupus.

Detection of autoantibodies to tumour-associated antigens in sera of patients with systemic autoimmunity using a novel protein microblot array.

It is well known that sera of patients with systemic autoimmunity contain autoantibodies to nuclear antigens. It is also known that patients with systemic autoimmunity have an increased risk for the development of tumours. Interestingly, tumour patients frequently develop autoantibodies and there is a growing list of potential tumour-associated antigens. It is, however, not known whether or not patients with systemic autoimmunity also develop antibodies to tumour-associated antigens. Here we describe the development of a novel multiprotein array allowing us to screen for autoantibodies to 30 different tumour-associated antigens in parallel. Using this novel assay, we found that the frequency of autoantibodies to the selected tumour-associated antigens is increased between 2- and 14-fold in patients with systemic autoimmunity compared with an age-matched control group.

The effects of previous hysterectomy on lupus.

Hysterectomy is one of the most common surgical procedures performed in United States, and currently, one in three women in United States has had a hysterectomy by the age of 60 years. Systemic lupus erythematosus (SLE) is a common autoimmune disease and especially targets women of childbearing age at least 10 times higher than men, which reflects the major role of female sex hormones. In this retrospective study, we evaluate the potential effects of previous hysterectomy in our lupus cohort. Data collected from study subject questionnaires were obtained from the Lupus Family Registry and Repository (LFRR) at the Oklahoma Medical Research Foundation. Hysterectomy data were available from 3389 subjects. SLE patients with a positive history of hysterectomy have been selected and compared with matched lupus patients with a negative history of hysterectomy and healthy controls. Association analyses were performed, and the P values and adjusted odds ratios (ORs) were calculated. SLE patients with a negative history of hysterectomy more likely had kidney nephritis or positive anti-dsDNA than age-matched SLE patients with a history of hysterectomy before disease onset. This effect was independent of ethnicity with an OR of 6.66 (95% CI = 3.09-14.38, P = 1.00 x 10(-8)) in European patients and 2.74 (95% CI = 1.43-5.25, P = 0.001) in African-Americans. SLE patients with a positive history of hysterectomy before disease onset also had a later age of disease onset (P = 0.0001) after adjustment for age and race. Our findings support the notion that the influence of female sex hormones in SLE and various clinical findings are tremendous and that surgical menopause such as this could significantly affect the outcome of disease and clinical manifestations.

Oral manifestations of Sjögren's syndrome.

Sjögren's syndrome is a common autoimmune rheumatic disease. The most common symptoms of Sjögren's syndrome are extreme tiredness, along with dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia). Saliva plays an essential role in numerous functions of the mouth. Xerostomia can be caused by medications, chronic diseases like Sjögren's syndrome, and medical treatments, such as radiation therapy and bone marrow transplant. Xerostomia can eventually lead to difficulty in swallowing, severe and progressive tooth decay, or oral infections. Despite having excellent oral hygiene, individuals with Sjögren's syndrome have elevated levels of dental caries, along with the loss of many teeth, early in the disease. Sjögren's syndrome alters the protein profile and brings about a change in the composition of saliva. There is an increase in the levels of lactoferrin, beta(2)-microglobulin, sodium, lysozyme C, and cystatin C, and a decrease in salivary amylase and carbonic anhydrase. Up to 90% of individuals with Sjögren's syndrome have antibodies targeting the Ro 60 and La autoantigens. Natural aging, regardless of Sjögren's syndrome, is also another factor that brings about a significant change in the composition of saliva. The most prevailing cause of xerostomia in elderly persons is the use of anticholinergic medications. Currently, there is no cure for Sjögren's syndrome, and treatment is mainly palliative.

Protein-protein interaction of the Ro-ribonucleoprotein particle using multiple antigenic peptides.

Protein protein interactions play a significant role in maintaining the structural and functional integrity of the cell. We used multiple antigen peptides (MAPs) to analyze such interactions within the Ro (or SSA) ribonucleoprotein complex. Our data showed that 60 kD Ro and La colocalize in the nucleus of the cell. Previous data have indicated that 60 kD Ro and La co-exist via interactions with the hYRNAs. We were interested to see whether 60 kD Ro and La interact with each other through protein protein interactions. MAPs were produced with sequences derived from the autoepitopes of 60 kD Ro. When used in agarose immunodiffusion certain MAPs formed precipitin lines specifically with Ro and La antigens. Used in affinity chromatography the Ro MAPs purified the Ro ribonucleoprotein particle from lymphocyte extract. Solid phase immunoassay and surface plasmon resonance (SPR) confirmed the observations obtained with agarose diffusion. Using SPR, kinetic analyses gave an apparent affinity constant of about 1 x 10(7) M(-1) for Ro-MAP-60 kD Ro interactions. The autoantigens Ro and La are specific targets in autoimmune diseases, particularly systemic lupus erythematosus (SLE) and Sjögren's syndrome, and are known to exist together as a complex with hYRNAs. The present data indicate that there are protein-protein interactions between Ro and La.

Presence of anti-La(SS-B) is associated with binding to the 13-kD carboxyl terminus of 60-kD Ro(SS-A) in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE) clinical manifestations, autoantibody production, and immunogenetics are inter-related. The ability to study parts of the autoimmune response may allow a more detailed understanding of these relationships. We undertook this study to determine whether the fine specificity of the autoimmune response to 60-kD Ro(SS-A) was related to the presence of other autoantibodies. We screened 74 patients with SLE for antibodies to the carboxyl 13-kD terminal of 60-kD Ro(SS-A) (13 kD). Twenty-five sera had such antibodies. This reactivity was distinguished by the presence of not only anti-Ro(SS-A) but also other antibodies. All nine sera with Ro(SS-A) and La(SS-B) Ouchterlony immunodiffusion precipitins bound 13-kD (p = 0.01), whereas 10 of 11 sera with both anti-Ro(SS-A) and anti-La(SS-B) as determined by immunosorbent assay bound 13-kD (p = 0.002). Inhibition studies demonstrated that antibodies binding the 13-kD fragment bound the 60-kD Ro(SS-A) protein but did not bind the La(SS-B) protein. Thus, anti-La(SS-B) was found in those sera that bound epitopes within the 13-kD carboxyl terminal of 60-kD Ro(SS-A). These data suggest a structural basis by which anti-Ro(SS-A) and anti-La(SS-B) are coupled in SLE.

Systemic lupus erythematosus: considerations for a genetic approach.

There is a powerful evidence suggesting that etiology and pathogenesis of systemic lupus erythematosus has both genetic and environmental components. Unfortunately, understanding the genetics of lupus has been impeded by knowing the pattern of inheritance. Indeed, a complex mode of inheritance for the lupus disease phenotype is suggested by the known characteristics of this disorder. Twenty-five multiplex pedigrees for lupus have been enrolled and have been used to evaluate power to reveal linkage. The power to find linkage in these pedigrees is greater for autosomal recessive than for autosomal dominant modes of inheritance. Once 100 similar pedigrees are available for analysis our results predict that linkage is likely to be present for genetic models with relatively relaxed requirements. At loci operating by autosomal recessive inheritance linkage should be detectable despite genetic homogeneity as low as 40% and penetrance as low as 50%. For loci operating by autosomal dominant inheritance genetic homogeneity must be 60% or more when penetrance is as low as 50% to be able to establish linkage. Available preliminary data are also consistent with a possible genetic linkage of Fc gamma RIIIPMN with lupus in American Black pedigrees multiplex for lupus.

Milk-alkali syndrome associated with calcium carbonate consumption. Report of 7 patients with parathyroid hormone levels and an estimate of prevalence among patients hospitalized with hypercalcemia.

Milk-alkali syndrome can be caused by ingesting large amounts of calcium carbonate. Coincident with the promotion of calcium carbonate as treatment for both dyspepsia and osteoporosis, milk-alkali syndrome is now a common cause of hypercalcemia severe enough to require admission to the hospital. The syndrome accounted for less than 2% of such admissions before 1990, but from 1990 through 1993, it was the cause of hypercalcemia for over 12% of these patients. Only primary hyperparathyroidism and hypercalcemia of malignancy (excluding multiple myeloma) are more common. The diagnosis of milk-alkali syndrome is made almost entirely based on the patient's history; careful attention to dietary practices and over-the-counter drug use is required, as numerous over-the-counter medications contain calcium carbonate. Modern assays for PTH demonstrate the expected suppression of PTH by hypercalcemia. Nonetheless, measurement of PTH must be performed in a timely manner as treatment with intravenous saline may result in hypocalcemia and elevated PTH soon after admission. Given the pathophysiology of milk-alkali syndrome compared to other causes of hypercalcemia, hypocalcemia with rebound hyperparathyroidism is probably unique to milk-alkali syndrome.

Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens.

Western blotting is a very sensitive and powerful fundamental technique in immunology that has been used to detect and characterize proteins of low abundance. This technique employs the transfer of proteins separated on SDS-PAGE to nitrocellulose sheets for further detection using antibodies. Here we report the non-electrophoretic transfer of the 60-kDa Ro (or SSA) autoantigen, 240 and 220 kDa spectrin antigens and prestained molecular weight standards from SDS-PAGE gels to nitrocellulose to obtain multiple immunoblots. In fact, we have used this procedure to obtain 12 immunoblots from a single gel with multiple sera.