Prevalence of 60 kda and 52 kda Ro/SS-A autoantibodies in anti-Ro positive Thai sera in Siriraj Hospital.

BACKGROUND: Anti-Ro antibody may directly react against either Ro60 or Ro52 or both antigens. To be more applicable for routine laboratory practice, the specific antigen type for antibody detection should be identified before test application. OBJECTIVE: Investigate the prevalence of 60 kDa and 52 kDa Ro/SS-A antibodies in Thai patients' sera in Siriraj Hospital. MATERIAL AND METHOD: Specimens for anti-Ro were requested between June and December 2005. They were tested with EUROLINE test kit for prevalence determination. The principle of the test is a qualitative in-vitro-assay that contains test strips coated with parallel lines of 14 highly purified antigens. Of 84 specimens requested for anti-Ro antibody, 76 were collected and tested with the EUROLINE test kits and eight were excluded due to inadequacy. RESULTS: The prevalence of anti-Ro60 and anti-Ro52 of all sera tested for anti-Ro by EUROLINE test kit were 30% (95% CI: 20-40%) and 26% (95% CI: 16-36%), respectively; and, those in anti-Ro positive Thai sera were 82% (95% CI: 68-96%) and 71% (95% CI: 54-88%), respectively. The prevalence of anti-Ro52 alone in anti-Ro positive Thai sera and all specimens requested for anti-Ro was about 18% (95% CI: 4-32%) and 7% (95% CI: 1-13%), respectively. The agreement and Kappa value between the two methods were 0.9 and 0.77, respectively. The study suggests that the test for anti-Ro detection should provide both Ro 60 and Ro 52 antigens. CONCLUSION: The prevalence of both anti-Ro 60 and anti-Ro 52 were quite common, therefore, the test for this specific antibody should provide both antigens for antibody detection.

Effect of a hypothetical gene Af116609 on multi-drug resistance of gastric cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR.</p><p><b>METHODS</b>Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells.</p><p><b>RESULTS</b>The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees.</p><p><b>CONCLUSION</b>Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.</p>

Transfection, overexpression and clinical application of human 60 kDa Ro/SSA autoantigens in HEp-2 cells / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.</p><p><b>METHODS</b>60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.</p><p><b>RESULTS</b>Stable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight over ten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60.</p><p><b>CONCLUSIONS</b>As a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.</p>

Antibodies to Ro/SS-A and La/SS-B in systemic lupus erythematosus and other autoimmune disorders.

AIM: 1. To study the presence of anti-Ro/SS-A, anti-La/SS-B, anti-Sm and anti-nRNP in diagnosed antinuclear factor (ANF) positive systemic lupus erythematosus (SLE) cases and their association with various organ involvement. 2. To study autoantibodies in other autoimmune disorders. MATERIAL AND METHODS: A total of 4050 suspected cases of autoimmune disorders referred for serological work up were evaluated for ANF by indirect immunofluorescence technique, anti-dsDNA by PHA, autoantibodies to Ro-SS-A and La/SS-B by ELISA and rheumatoid factor was tested by latex agglutination using commercial kits. RESULTS: Out of 4050 patients 19.5% were ANF positive and 5% were anti-dsDNA positive. Out of these 50 diagnosed ANF positive cases of SLE, an incidence of anti-dsDNA 54%, anti-Sm 25.9%, anti-nRNP 29.6%, anti-Ro/SS-A 10% and anti-La/SS-B was 22% was observed. In rheumatoid arthritis, 17.4% positivity of anti-Ro/SS-A and 39.1% positivity for anti-La/SS-B was observed. In SLE with renal involvement, joint complaints and skin or malar rash were seen in 66%, 56% and 46%, respectively. CONCLUSION: Determining anti-Ro/SS-A and anti-La/SS-B antibody could be important in evaluating patients with suspected connective tissue disorders, who usually show diverse clinical presentations like skin, kidney and joint manifestations. The most prominent feature in anti-Ro/SS-A and anti-La/SS-B positive patients was skin involvement and sicca complex in 60% of SLE patients.

Comparison of counter immunoelectrophoresis with immunoblotting for detection of anti-extractable nuclear antigen antibodies in systemic lupus erythematosus.

Anti-extractable nuclear antigen (ENA) antibodies were assayed by counter immunoelectrophoresis (CIE) and immunoblotting in patients with systemic lupus erythematosus (SLE). We found the two methods showed good concordance rates, the lowest being 67% for anti-SS-A. Immunoblotting was more sensitive in detecting anti-Sm, anti-SS-B and anti-PCNA (proliferating cell nuclear antigen); CIE was more sensitive for anti-nRNP and anti-SS-A. Overall, the prevalence of these anti-ENA antibodies in SLE was increased by 9-20% if immunoblotting was used in addition to CIE. Sera specific for the 52 kDa peptide of the SS-A antigen (anti-52kDa SS-A) were better detected by immunoblotting. Anti-PCNA antibody was found in 6.3% of SLE patients and was associated with active disease and hemolytic anemia. The positive rate of anti-Sm was 9% by CIE and 23.7% by immunoblotting and this antibody was a specific marker for SLE using either method. It was concluded that using immunoblotting in addition to CIE, the overall sensitivity of detection of anti-ENA antibodies in SLE was increased and clinically useful antibodies such as anti-52kDa SS-A and anti-PCNA could be detected.