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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Autoantibody determination in the diagnosis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that usually develops in young women aged 18-50 years and is characterized by the presence of autoantibodies. Diagnosis is difficult as SLE is a great imitator of other diseases. When SLE is suspected clinically in a patient (involvement of two or more organ systems), an initial laboratory evaluation would be antinuclear antibody (ANA) testing. If ANA is negative, SLE is unlikely and results positive at less than 1:40 strongly argue against SLE. Other explanations for organ system involvement should be pursued. Results positive at greater than 1:40 may merit further evaluation for SLE and at times referral to a rheumatologist for a full SLE evaluation. While the American College of Rheumatology classification criteria for SLE are primarily a tool for research, they may be useful clinically, in that those patients fulfilling four or more criteria are highly likely to have SLE.

Induction of oral tolerance in experimental Sjogren's syndrome autoimmunity.

Previous studies have showed that immunization with peptides from Ro 60 results in Sjogren's syndrome (SS)-like condition in BALB/c mice. We hypothesized that oral feeding with Ro 60 peptide or Ro 60 would prevent the disease. Four groups (each consisting of 10) of BALB/c mice were used. Group I-III were immunized with Ro 274 peptide. Group IV mice were administered adjuvant only. Group II mice were fed orally with Ro 274 peptide and Group III with Ro 60 for 5 days before immunization. There was a significant reduction in the binding of sera from both Group II and Group III mice to most of the Ro multiple antigenic peptides bound by Group I mice. In Group III mice, salivary flow was maintained above that of the Group I mice (average: 117.5 versus 58.6 microl; t = 2.7; P = 0.02). Salivary infiltrates were drastically decreased in the Ro peptide or Ro 60-fed groups, compared to non-tolerized group. Two of eight mice in Group II and 3/6 mice in Group III had no infiltrates, whereas all eight mice studied in Group I had a significant number of infiltrates. Thus, epitope spreading was prevented, lymphocytic infiltration was blocked and saliva flow was restored by means of oral feeding of either Ro 274 or Ro 60 in this animal model of SS.

Toxic epidermal necrolysis-like acute cutaneous lupus erythematosus and the spectrum of the acute syndrome of apoptotic pan-epidermolysis (ASAP): a case report, concept review and proposal for new classification of lupus erythematosus vesiculobullous skin lesions.

The acute clinical syndrome of toxic epidermal necrolysis (TEN) is currently thought to be a distinct clinical-pathological entity typically resulting from drug hypersensitivity. We describe an adult woman who experienced a fulminate pattern of apoptotic epidermal cell injury following tanning bed exposure while taking naproxen that resulted in a clinical presentation having combined features of drug-induced TEN and an infrequently recognized form of bullous cutaneous lupus erythematosus (LE). This case calls attention to the fact that TEN-like injury can occasionally be seen in settings other than drug hypersensitivity (e.g., LE, acute graft versus host disease) and illustrates the need for a unifying concept in this area. We therefore propose the term 'Acute Syndrome of Apoptotic Pan-Epidermolysis (ASAP)' to designate a clinical syndrome that is characterized by life-threatening acute and massive cleavage of the epidermis resulting from hyperacute apoptotic injury of the epidermis. We also review vesiculobullous skin disorders that can be encountered in LE patients and suggest a new classification scheme for such lesions.

Immunization with peptides from 60 kDa Ro in diverse mouse strains.

Antibodies binding the Ro (or SSA) and La (or SBB) proteins are commonly found in a high proportion of sera from patients with systemic lupus erythematosus or Sjögren's syndrome. The mechanism by which these autoantibodies arise is not known. Others and we have shown that immunization of nonautoimmune-prone mice with short peptides from the Ro ribonucleoprotein particle can induce autoimmunity to 60 kDa Ro and 52 kDa Ro as well as to the 48 kDa La protein after epitope spreading. We have explored the differences in the epitope spreading after 60 kDa Ro peptide immunization in several strains of mice. There is intra- and intermolecular diversification of the immune response after immunization of DBA/2J animals with a monomer peptide representing the residues 480-494 of the 60 kDa Ro protein, but this peptide does not induce epitope spreading when used as the immunogen in either C57Bl/6J or PL/J mice. Similar to previously studied BALB/c mice, DBA/2J mice have antibodies binding many epitopes of 60 kDa Ro, and some sera bind 52 kDa Ro as well as La. These mice have antinuclear antibody in their sera. These data demonstrate that Ro peptide immunization results in different outcomes depending upon the strain of mouse used. Furthermore, these data suggest that genetic variation is important with regard to responding towards short peptide immunization by epitope spreading.

Selective small antigenic structures are capable of inducing widespread autoimmunity which closely mimics the humoral fine specificity of human SLE.

Recent data have suggested that autoantibodies in lupus can progress from simple immunity against a few antigenic structures to a complex response against multiple autoantigens. Our aim was to determine whether these diverse epitope patterns can indeed be generated by antigenic challenge with a single, small structure. Rabbits were immunized with either a 60 kDa Ro peptide commonly antigenic in human systemic lupus erythematosus (SLE) (Ro 274-289) or one which is rarely a humoral target (Ro 500-515). Rabbits immunized with the antigenic peptide (Ro 274-289) not only developed antibodies to multiple epitopes of 60 kDa Ro and La, as has been described, but also produced non-cross-reactive antibodies to the common spliceosomal proteins Sm B' and D1, and nRNP A and C. Rabbits immunized with the Ro 274-289 peptide also mount a progressive, diversified immune response to the sequential antigenic regions of these proteins (60 kDa Ro, Sm B' and D1, nRNP A and C), which is nearly identical to that seen in human SLE. Animals immunized with the nonantigenic peptide Ro 500-515 develop antibodies only to 60 kDa Ro. These results demonstrate that loss of tolerance to select single, small antigenic structures can begin a cascade which virtually recreates, at the epitope level, the humoral autoimmune specificity seen in human SLE.