Sputum Autoantibodies Are More Relevant in Autoimmune Responses in Asthma than Are Serum Autoantibodies

PURPOSE: The data on the differences between sputum autoantibodies (Sp-Abs) and serum autoantibodies (Se-Abs) in reflection of autoimmune responses to lungs is still lacking. METHODS: Ten types of Abs were investigated in matched Se and Sp samples collected from recruited subjects. Correlations between Ab levels and airway inflammatory parameters and measures of pulmonary function were assessed. The network-based and inter-correlated analysis was performed to explore the patterns of Sp- and Se-Ab profiles. RESULTS: Fifty stable asthmatic patients and 24 healthy volunteers were recruited for our study, 15 with mild asthma, 18 with moderate asthma and 17 with severe asthma. The concentrations of Sp-Ab against U1 small nuclear ribonucleoprotein (Sp-anti-U1-SnRNP), Sp-Ab against Smith antigen and Se-Ab against thyroid peroxidase (anti-TPO) in severe asthmatics and Sp-anti-U1-SnRNP in moderate asthmatics were significantly higher compared to healthy controls and mild asthmatic subjects (P < 0.05). Sp-anti-U1-SnRNP levels were positively correlated with the dose of inhaled corticosteroids, Sp eosinophil counts and fractional exhaled nitric oxide (r = 0.326, P = 0.022; r = 0.356, P = 0.012; r = 0.241, P = 0.025, respectively) and negatively correlated with Sp neutrophil counts (r = −0.308, P = 0.031) with adjustment for age. Spearman's correlation matrix showed multiple inter-correlations among Sp-Abs and Se-Abs (P < 0.05) while only the levels of Ab against DNA topoisomerase and anti-TPO in Se were correlated with those Sp-Ab counterparts (P < 0.05). The network-based analysis defined 2 clusters: clusters 1 and 2 contained 10 Sp-Abs and 10 Se-Abs, respectively. CONCLUSIONS: This study observes that Sp-Abs are more associated with clinical parameters and the severity of disease in asthma compared to Se-Abs. Targeting on Sp-Abs which are the hallmark of the localized autoimmune event might help us better understand the role of autoimmunity in the pathological mechanism of asthma.

Association of microRNA-related genes (DROSHA, DICER1 and GEMIN4) polymorphisms with T-cell lymphoma prognosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the association of micoRNA-related genes DROSHA single nucleotide polymorphisms (SNP) rs10719 and rs6877842, DICER1 rs3742330and GEMIN4 rs3744741 with prognosis of T-cell lymphoma.</p><p><b>METHODS</b>Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the genotypes of the above 4SNPs and their associations with complete remission (CR) rate and overall survival (OS) in 163 patients with TCL.</p><p><b>RESULTS</b>Patients carrying the rs6877842 CG genotype had a significantly higher CR rate compared with those carrying the CC genotype (OR=0.07, 95% CI 0.01-0.72, P=0.026); the same for patients carrying the DICER1 rs3742330 GG genotype compared with those carrying the GA genotype (OR=0.15, 95% CI 0.02-0.97, P=0.047) or the AA genotype (OR=0.11, 95% CI 0.02-0.71, P=0.020). In addition, patients with the DICER1 rs3742330 GG genotype had a significantly improved OS compared with those carrying the GA (HR=9.02, 95% CI 1.22-66.92, P=0.031) or AA genotype (HR=8.77, 95% CI 1.19-64.67, P=0.033). The other two SNPs of rs10719 and rs3744741 had no significant association with CR or OS.</p><p><b>CONCLUSION</b>DROSHA rs6877842 and DICER1 rs3742330 were independent factors for TCL CR, and DICER1 rs3742330 was also an independent prognostic factor for TCL OS.</p>

Spliceosomal genes in the D. discoideum genome: a comparison with those in H. sapiens, D. melanogaster, A. thaliana and S. cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D. discoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.

Pseudouridines in spliceosomal snRNAs

Spliceosomal RNAs are a family of small nuclear RNAs (snRNAs) that are essential for pre-mRNA splicing. All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription. Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing. Many of these modified nucleotides are conserved across species lines. Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms: an RNA-dependent mechanism and an RNA-independent mechanism. The functions of the pseudouridines in spliceosomal snRNAs (U2 snRNA in particular) have also been extensively studied. Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important. Besides the currently known pseudouridines (constitutive modifications), recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions, thus strongly suggesting that pseudouridylation is also a regulatory modification.

High serum levels of rheumatoid factor and anti-phosphatidylserine antibody in patients with ischemic heart disease

Immunopathological and inflammatory processes play important roles in the initiation and development of Ischemic Heart Disease [IHD]. The aim of this study was to evaluate the serum levels of several autoantibodies including rheumatoid factor [RF], anti-nuclear antibodies [ANA], anti-small nuclear ribonucleoprotein [anti-Sm], anti-phosphatidylserine [anti-PS] and anti-cardiolipin [anti-CL] antibodies in patients with IHD. A total of 120 patients with IHD with acute myocardial infarction [AMI; n=60] or unstable angina [UA; n=60] and 60 sex- and age-matched healthy subjects were enrolled in this study. Serum samples of participants were tested for the ANA, anti-Sm, anti-PS and anti-CL antibodies by ELISA. Serum level of RF was measured by a turbidometric method. The mean serum levels of RF and anti-PS antibodies in AMI group and UA group were significantly higher than those observed in the control group [p<0.0001]. The mean serum levels of RF and anti-PS antibodies in AMI patients were significantly higher than the UA group [p<0.01 and p<0.001, respectively]. The mean serum levels of RF in men with AMI or UA diseases were significantly higher as compared to healthy control men [p<0.0001 and p<0.003, respectively]. The differences of the serum levels of ANA, anti-Sm and anti-CL antibodies were not significant between AMI, UA and the control groups. There was no difference in the serum levels of RF, ANA, anti-Sm, anti-PS or anti-CL antibodies in patients with traditional risk factors, including hypertension, dyslipidemia, diabetes and smoking, and those without a certain risk factor. Higher serum levels of RF and anti-PS antibody in patients with IHD may be considered as independent risk factors for IHD

Methylation Patterns of Small Nuclear Ribonucleoprotein Polypeptide N (SNRPN) Related to the Germ Cell Differentiation of Human Germ Cell Tumors

BACKGROUND: The histogenesis and interrelationship of the various types of germ cell tumors (GCTs) have been proposed. Dysgerminoma/seminoma (D/S) is a primitive GCT that has not acquired the potential for further differentiation, whereas other types of GCTs are in a dynamic process of differentiation towards a somatic or extraembryonal direction. A primordial germ cell giving rise to a GCT undergoes a developmentally regulated erasure and resetting of imprinted genes, but changes in the imprinting pattern in GCTs as the tumor differentiates have not been well defined. We aimed to investigate the changes of the SNRPN methylation pattern between the germinomas and non-germinomatous GCTs, as compared with the somatic methylation pattern. METHODS: We used formalin-fixed paraffin-embedded tissue sections of 97 GCTs (18 Ds, 21 Ss, 17 yolk sac tumors (YSTs), 19 immature teratomas, and 22 mature teratomas). DNA methylation was evaluated after bisulfite modification, PCR amplification, and restriction enzyme digestion. RESULTS: The SNRPN methylation pattern was changed in 53/74 (71.6%) of GCTs as non-somatic patterns. There were significant differences in the methylation pattern between the germinomas and non-germinomatous GCTs, the GCTs being frequently hypo- methylated in Ds/Ss (73.3%), in contrast to the frequent hypermethylation seen in the YSTs and teratomas (47.7%, p<0.05). CONCLUSIONS: The methylation status of an imprinting gene may be involved in the mechanism causing cellular differentiation and tumorigenesis of GCTs.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95 percent of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31 percent reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Molecular markers for diagnosis of Prader-Willi syndrome in thai patients by fish.

Paternal microdeletion of chromosome 15 at q11-q13 has been reported in 75% of Prader-Willi syndrome (PWS) patients in western countries. Diagnosis of PWS in Thailand is mainly based on clinical observation and, in some cases, confirmed by conventional cytogenetic analysis. Loss of a tiny segment in this region (microdeletion) has made it difficult to discriminate from the normal karyotype. An attempt to solve this problem has been made by using a high resolution chromosome culture. However, this method is a tedious and time-consuming technique which is suitable for only experienced cytogeneticists. We report molecular cytogenetic analysis for PWS in Thai patients using FISH in addition to standard GTG- banding chromosome analysis. Nine Thai patients clinically diagnosed or with a suspicion of PWS were investigated. The FISH probes consist of the region-specific probes (SNRPN or D15S10 probe) and two chromosome 15-specific control probes (D15Z1 centromeric and PML chromosome 15 long arm probe). Bright field and FISH programs of an automatic karyotyper were applied to facilitate the efficiency of the chromosome analysis. We found that 2 out of 9 patients showed a deletion at 15q11-q13 region by standard GTG chromosome analysis while 4 out of 9 patients showed a delation in this region by FISH. Consistent losing of SNRPN and D15S10 signals in FISH was observed in these patients. This forty-four per cent deletion is considerably lower than those reported from western countries. We propose that DNA methylation at SNRPN promoter as well as structural abnormalities in other chromosome regions might also play a role in the etiology of this disorder in Thais, which should be investigated further.

Advance in researches on survival of motor neurons protein and its interaction proteins / 中华医学遗传学杂志

Survival of motor neurons(SMN) protein is the product of spinal muscular atrophy(SMA) gene. Now the function researching of SMN protein has become hotspot field to discuss the pathogenic mechanism of SMA. The construction, distribution and function of SMN protein are reviewed in this paper.

Prader-Willi syndrome and genomic imprinting / 中华儿科杂志

<p><b>OBJECTIVE</b>Prader-Willi syndrome (PWS) is an example of a human genetic disorder that involves imprinting genes on the proximal long arm of chromosome 15 and SNRPN gene as a candidate gene for this syndrome. The purpose of this study was to show the molecular genetic defects and genomic imprinting basis in Chinese PWS patients and to evaluate the clinical applications of a differential diagnostic test for PWS.</p><p><b>METHODS</b>Fluorescence in situ hybridization (FISH) and methylation-specific PCR (MSPCR) techniques were applied for 4 clinically suspected PWS patients. Using three probes, including SNRPN probe for identification of the critical locus in PWS region, D15Z1 and PML control probes for identification of the 15p arm and 15q arm, the authors detected the deletions 15q in PWS. MSPCR was based on sodium bisulfite treatment of DNA and PCR primers specific for the maternal and paternal allele.</p><p><b>RESULTS</b>When hybridized with mixed probes, it was found in 2 patients that the central specific signal was absent, but both the flanking control signals were retained, indicating SNRPN gene deletion of chromosome 15q11-13. Bisulfite-modified DNA from all PWS children amplified with methylated allele-specific primer pair showed only maternal 131bp PCR product, indicating the maternal uniparental disomy (UPD15).</p><p><b>CONCLUSION</b>Genomic imprinting plays an important role in the molecular pathogenesis of PWS that caused by paternal microdeletions of 15q11-q13 or maternal UPD of chromosome 15. The basic defect seemed to be an absence of function of PWS genes that are normally expressed only from the paternal chromosome 15. MSPCR is a rapid and simple PCR-based assay compared with other cyto-molecular tests and its results were consistent with the clinical diagnosis of PWS, so it seems to be a reliable diagnostic method for PWS patients who show abnormal methylation at SNRPN. The genetic differential tests for PWS are important in determining familial recurrence risk.</p>

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.

Molecular Diagnostic Test for Prader-Willi Syndrome with SNRPN Expression / 대한소아내분비학회지

PURPOSE: Prader-Willi Syndrome(PWS) is caused by absence of paternal contributions of the chromosome region 15q11-q13. To detact this region, high resolutional cytogenetic analysis, FISH with probe at PWS critical region or microsatellite polymorphism can be used. The gene for the small nuclear ribonucleoprotein polypeptide N(SNRPN) is not expressed in patients with PWS. We conducted molecular analysis with RT-PCR with SNRPN primers to find out more useful diagnostic tool in PWS. METHODS: Four patients with obesity and other characteristics of PWS were studied. The exprssion of SNRPN and control gene were studed by RT-PCR from peripheral lymphocytes. RESULTS :The SNRPN expression in reverse transcribed RNA from blood were easily detected in normal control but not in patients with suspected Parder-Willi Syndrome. CONCLUSION: We conclude that SNRPN expression study is a useful diagnostic method for detection of Prader-Willi Syndrome.

The prevalence of antinuclear, anti-dsDNA, anti-Sm and anti-RNP antibodies in a group of healthy blood donors.

We studied the prevalence of antinuclear (ANA), anti-double stranded DNA (dsDNA), anti-Sm and anti-RNP antibodies in a group of 93 blood donors (age range: 18-58 years). Antinuclear and anti-ds DNA antibodies were detected by immunofluorescence (IF) using HEp2 cells and Crithidia luciliae as substrates, respectively, while anti-Sm and anti-RNP antibodies were assayed by ELISA. ANA was found in 6.5% while anti-dsDNA antibodies were not detected in any of the subjects. The 98th percentile was used as cut off where values greater than 0.651 for anti-Sm and 0.601 for anti-RNP antibodies were taken to be positive. This gives a frequency of 1.1% for both antibodies. There was no significant association of antibody positivity with sex or race. We conclude that certain autoantibodies are present in low titres in the normal Malaysian Individuals, at a different frequency compared to other studies probably due to genetic, ethic or environmental factors.