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.

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.

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

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.

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.

Polyethylene glycol-mediated bacterial colony transformation.

A moderately efficient and quick method of bacterial colony transformation is described. Plasmid DNA was added to bacteria suspended in a solution of polyethylene glycol/calcium chloride (PEG/CaCl2). After a brief incubation and heat shock, the cells were directly plated. Transformation efficiencies up to 8.6 +/- 1.28 x 10(6) transformants per microgram of pUC18 were obtained. We have found that the reverse of the transformation process could also take place. Suspending a bacterial pellet harboring the plasmid of interest in PEG/CaCl2 results in the release of the plasmid DNA, and thus indirectly lends support to the transformation process.

Rheumatoid hyperviscosity: analysis of a patient with intermediate complexes that block other autoantibodies and a review of the literature.

OBJECTIVES: To report a patient who rheumatoid arthritis presented with hyperviscosity syndrome, analyze this patient's rheumatoid factor, and review the previously reported patients. METHODS: Immunofluorescence for antinuclear antibodies, double immunodiffusion, enzyme-linked immunosorbent assay, and size exclusion chromatography were used before and after plasmapheresis to study the patient's rheumatoid hyperviscosity, and polyclonal gammopathy and references in identified papers was used to identify previously reported patients. RESULTS: Similar to several previous patients, this patient's sera contained both IgG and IgM rheumatoid factor and abundant intermediate complexes. Other autoantibodies, either from the patient or from other patients, were masked by rheumatoid factor or intermediate complexes from the reported patients sera. Rheumatic hyperviscosity is seen uncommonly, being reported in only 18 patients with rheumatoid arthritis and nine with other rheumatic illnesses. CONCLUSIONS: There are two mechanisms by which rheumatoid factor can lead to hyperviscosity, both of which require large amounts of rheumatoid factor. Rheumatoid hyperviscosity must be recognized because this life-threatening syndrome usually can be successfully treated with plasmapheresis acutely and immunosuppressives for long-term control.

Immunization with vesicular stomatitis virus nucleocapsid protein induces autoantibodies to the 60 kD Ro ribonucleoprotein particle.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder of unknown etiology that is characterized by antibodies binding ribonucleoprotein complexes. The epitopes of SLE associated 60 kd Ro (or SSA) autoantigen share sequence with vesicular stomatitis virus (VSV) nucleocapsid (N) protein and SLE patients with anti-Ro are more likely to bind the N-protein than anti-Ro negative patients. METHOD: We immunized New Zealand white (NZW) rabbits with purified VSV N-protein to examine the relationship between the immune response to N-protein and anti-Ro. RESULTS: After multiple immunizations with VSV N-protein, NZW rabbits produced not only IgG antibodies to VSV N-protein but also an autoimmune response to Ro antigen. The IgG fraction of the rabbit immune serum precipitates 60 kd Ro protein as well as its associated Y RNAs. Antibodies elicited by 10 immunizations of VSV N-protein bind to at least 20 linear epitope groups on VSV N-protein and over 25 linear epitope groups on 60 kd Ro protein. These antibodies also bound 5 of the 6 shared sequences between 60 kd Ro protein and VSV N-protein. CONCLUSIONS: Our data demonstrate that an immune response initiated towards a foreign antigen, selected on the basis of sharing short sequence similarity with the autoantigenic epitopes of an autoantigen, can lead to an autoimmune response.

Mouse urine collection using clear plastic wrap.

Qualitative urinalysis using Multistix reagent strips for the detection of urinary pH, protein, glucose, bilirubin, blood, ketone, urobilinogen and creatinine can be carried out with a few drops of mouse urine. The use of metabolic cages is not practical for such qualitative studies particularly when several animals are involved. Here we describe two different methods for collecting pure mouse urine. The single animal method (SAM) involves allowing a single mouse to urinate on Glad cling wrap outside of the animal cage. The multiple animal method (MAM) involves partitioning seven mice into seven different make-shift compartments laid out on top of the cling wrap and allowing them to urinate. The voided urine, in each case, is then aspirated into micro-centrifuge tubes using a Pipetman. Without coercion pure urine was obtained as early as 12 s. Volumes in the range of 10-250 microl were obtained. Modifications of the SAM could prove useful for rat or mouse urine collection under conditions of microgravity.

Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels.

For the detection of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Coomassie blue is used commonly on account of its simplicity and reliability. In this report we show that enhanced heat, in addition to dramatically decreasing the time required for staining and destaining, also significantly increased the detection sensitivity. For a 1.5 mm gel, the staining time was 5 min at 55, 62.5 or 70 degrees C while the destaining time was 45, 45 and 20 min respectively. For a 0.8 mm gel, the staining time could be reduced to 1 min at 65 degrees C compared to 2 min at 60 degrees C and 5 min at 55 degrees C. The destaining time required was 8, 15 and 20 min at the respective temperatures.

Multiple nuclear dot antinuclear antibody in patients without primary biliary cirrhosis.

Multiple nuclear dot (MND), or pseudocentromere, anti-nuclear antibody (ANA) is an uncommon pattern associated primarily with primary biliary cirrhosis (PBC) and anti-mitochondrial antibody (AMA). A 53 kDa antigen with an apparent molecular mass of 100 kDa as found on sodium dodecyl sulphate-polyacrylamide gel electrophoresis is thought to be responsible for the uncommon pattern. This study analyzes sera from 21 patients without PBC or AMA that produced the uncommon MND ANA immunofluorescence pattern. Diseases present include lupus, rheumatoid arthritis and scleroderma. On immunoblotting nineteen of 21 (91%) bound a 70 kDa protein. Western blot analysis showed that this nuclear antigen was different from pyruvate dehydrogenase, p80 coilin and the antigen responsible for MND ANA in those with PBC. Affinity purified anti-70 kDa reproduced the MND ANA immunofluorescence pattern. Thus, the MND ANA in patients without PBC/AMA is associated with binding to a 70 kDa nuclear protein and not with a 53 kDa antigen (that runs at 100 kDa) found in those with MND and PBC/AMA. The data demonstrate the MND antigen without PBC/AMA is immunologically distinct from the pattern when found with PBC/AMA.

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.

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.

Immunologically restricted and inhibitory anti-Ro/SSA in monozygotic twins.

A pair of monozygotic twins with Sjögren's syndrome are described who both have large amounts of anti-Ro/SSA in their sera by ELISA, but no precipitin in double immunodiffusion. Unique among previously encountered specimens, sera from the twins inhibit the formation of Ro/SSA-precipitins in double immunodiffusion by known anti-Ro/SSA positive SLE patients. The twins have near identical, clonally restricted anti-Ro/SSA autoantibodies as evaluated by isoelectric focusing and bind the same 60 kD Ro/SSA peptides. Thus, this pair of monozygotic twins has an identical fine specificity in their immune response to 60 kD Ro/SSA, despite potential differences in their immune response generated by random processes in the formation of immunoglobulin molecules and T cell receptors. These data imply that the anti-Ro/SSA found in these sera binds less than three epitopes such that soluble antigen/antibody complexes are formed instead of an insoluble complex that precipitates.