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B T Kurien

Publications and source records attributed to B T Kurien.

At least 19 recordsLinked to original sources
PubMed2006

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.

Antibodies, Antinuclear↗
PubMed2005

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.

Administration, Oral↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2001

Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction.

A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.

DNA↗
PubMed2001

Autoantibody to the leucine zipper region of 52 kDa Ro/SSA binds native 60 kDa Ro/SSA: identification of a tertiary epitope with components from 60 kDa Ro/SSA and 52 kDa Ro/SSA.

Anti-Ro (or SSA) is found in the sera of patients with autoimmune rheumatic illnesses. All patients with anti-Ro defined by precipitation bind a 60 000 Da antigen (60 kDa Ro), whereas some patients also bind a 52 000 Da molecule (52 kDa Ro). In general, antibody binding is directed against native 60 kDa Ro and denatured 52 kDa Ro. The mechanism by which anti-52 kDa Ro arises in the setting of anti-60 kDa Ro is unknown. Conflicting data exist as to the existence of a physical interaction between the two proteins in cells and as to cross-reacting antibodies. Antibodies were affinity purified from a peptide within the leucine zipper region of 52 kDa Ro. These purified antibodies binding the 197-207 peptide from 52 kDa Ro (anti-52LZ) bound native 60 kDa Ro as well as denatured 52 kDa Ro. In addition, anti-52LZ also bound up to four regions from the sequence of 60 kDa Ro and a single conformational epitope of 60 kDa Ro. Thus, these primary sites represent components of the tertiary epitope. We hypothesized that if this was the case, these peptides making up a tertiary epitope would show molecular interaction. In fact, peptides from 60 kDa Ro have a molecular interaction with the 52 kDa Ro peptide as well as full-length 52 kDa Ro when assessed by surface plasmon resonance. The leucine-zipper region peptide from 52 kDa Ro bound three of the four peptides from 60 kDa Ro. These data suggest that these two molecular species, 60 and 52 kDa Ro, form a conformational epitope. This relationship may explain why anti-52 kDa Ro is found in association with anti-60 kDa Ro.

Amino Acid Sequence↗
PubMed2001

Can B cell epitopes of 60 kDa Ro distinguish systemic lupus erythematosus from Sjögren's syndrome?

Antibodies binding components of the Ro/La (or SSA/SSB) ribonucleoprotein particle are found in the sera of patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) as well as mothers who give birth to babies with neonatal lupus. Anti-La occurs in a subset of sera that contain anti-Ro, and anti-La is found more commonly in sera of patients with SS than in sera from SLE patients. The fine specificity of autoantibodies binding 60 kDa has been studied extensively. Recent data have suggested that there are disease-specific epitopes which identify patients with either SLE or SS. Alternatively, other data suggest that the B cell epitopes of 60kDa Ro vary according to the presence of anti-La. The present study was undertaken to determine whether binding of putative disease-specific 60 kDa Ro epitopes is associated with the diagnosis of SLE vs SS, or instead associated with the presence of anti-La. Anti-60 kDa Ro positive sera from 24 SLE patients and 44 SS patients were studied for antibodies binding two epitopes of 60 kDa Ro. We find the epitope defined by residues 171-190 is associated with anti-60 kDa Ro without anti-La, regardless of diagnosis. Meanwhile, binding of the epitope defined by residues 215-232 is not commonly found in anti-60 kDa Ro sera, especially in those sera with both anti-60 kDa Ro and anti-La. Thus, the fine specificity of antibody binding to 60 kDa Ro varies according to the presence of anti-La, not to the diagnosis of either SLE or SS.

Autoantibodies↗
PubMed2001

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.

Humans↗
PubMed2000

Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen.

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.

Adult↗
PubMed2000

Anti-calreticulin segregates anti-Ro sera in systemic lupus erythematosus: anti-calreticulin is present in sera with anti-Ro alone but not in anti-Ro sera with anti-La or anti-ribonucleoprotein.

OBJECTIVE: To examine a well characterized group of patients with systemic lupus erythematosus (SLE) for anti-calreticulin. METHODS: The sera of 77 patients with SLE were studied by immunodiffusion, solid phase immunoassay, and immunoblot for antibodies against ribonucleoprotein (RNP) autoantigens and calreticulin. RESULTS: Thirty-five had anti-calreticulin and 40 had anti-60 kDa Ro. There was no association of anti-60 kDa Ro and anti-calreticulin. However, among anti-60 kDa Ro positive sera that also contained either anti-La or anti-RNP, none of 18 had anti-calreticulin. All the remaining sera with anti-60 kDa Ro had anti-calreticulin and anti-52 kDa Ro. CONCLUSION: Anti-60 kDa Ro patients with SLE can be divided into those with anti-60 kDa Ro and either anti-La or anti-RNP or those with anti-60 kDa Ro, anti-52 kDa Ro, and anti-calreticulin.

Autoantigens↗
PubMed1999

Immunization of mice with human 60-kd Ro peptides results in epitope spreading if the peptides are highly homologous between human and mouse.

OBJECTIVE: Immunization with peptide fragments of autoantigens may lead to an immune response at both the T and B cell level that is directed not only at the immunogen, but also at the autoantigen from which the peptide came. In addition, a complex multicomponent particle may become the target of this expanded immune response. The purpose of this study was to determine the ability of several different peptides from 60-kd Ro to induce expansion of the immune response to the Ro/La RNP particle. METHODS: We immunized BALB/c mice with 3 different oligopeptides from human 60-kd Ro (or, SSA). RESULTS: Animals immunized with peptides either identical to or differing by only 1 amino acid developed autoimmunity to the entire Ro RNP particle. Animals immunized with a human peptide highly divergent from the corresponding mouse sequence developed an immune response to the immunogen only and showed little evidence of epitope spreading. Furthermore, these mice did not have antibodies that bound the poorly conserved mouse homolog peptide, and the antibody response to this peptide did not include IgG1. CONCLUSION: These data indicate that B lymphocytes specific for the self-peptide that is homologous to the immunogen are a critical determinant for spreading of the immune response to other components of self.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

Immunoblotting of multiple antigenic peptides.

Multiple antigenic peptides (MAPs) can be efficiently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane for subsequent use in immunoblot (IgG and IgM). MAPs can be stained by Coomassie and silver on SDS-PAGE as well as by Fast Green on an immunoblot. Affinity immunoblotting for analysis of antibody clonotype distribution has also been carried out using MAPs. High performance liquid chromatography purification of the MAPs is mainly responsible for their migration as sharp bands in SDS-PAGE and immunoblotting.

Amino Acid Sequence↗
PubMed1998

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.

Adult↗
PubMed1998

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.

Coloring Agents↗
PubMed1998

Anti-centromere autoantibody in a patient evolving from a lupus/Sjögren's overlap to the CREST variant of scleroderma.

We characterized the development of the anti-centromere antibody in a patient prior to the development of CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) symptoms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting (IgG and IgM) of cellular extracts enriched for centromere antigens and indirect immunofluorescence were used to study the anti-centromere immune response. The sera recognized 3 centromere antigens with molecular masses 18,000 (CENP-A), 50,000 (CENP-D), and 80,000 (CENP-B). For CENP-A, IgM was present before the appearance of the IgG response. Anti-CENP-D revealed an IgM response that decreased over time but no IgG, while CENP-B showed an IgG response that strengthened and then weakened over time. The appearance of an anti-centromere nuclear fluorescence pattern correlated with the appearance of IgG anti-CENP-A. Signs and symptoms typical of CREST began about 4 years after antibodies to centromere antigens were found. The development of the CREST syndrome in our patient was preceded by the appearance of anti-centromere autoantibodies. For at least one of the antigens (CENP-A), there was an immunoglobulin class switch from IgM to IgG.

Adult↗