Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.     

Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates.

Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII. Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers. With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing. Twenty isolates grouped into six IB types were separated into seven major AP-PCR types. These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation). The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05. Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7. A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing. While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types. In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group. We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C. difficile.     

The complexity of HLA class II (DRB1, DQB1, DM) associations with disseminated Mycobacterium avium complex infection among HIV-1-seropositive whites

Earlier associations of polymorphism in classic HLA class II (DRB1 and DQB1) genes have been extended to include the accessory genes DMA and DMB as determinants of disseminated Mycobacterium avium complex (DMAC) infection among HIV-1-seropositive whites. From the Multicenter AIDS Cohort study, 176 DMAC cases were matched with 176 controls in a nested case-control study. PCR-based HLA genotyping techniques were used to resolve variants of DRB1 and DQB1 to their four-digit or five-digit alleles, and single-strand conformation polymorphism was used to resolve sequences in exon 3 at each DM locus. The DMA*0102 allele occurred less frequently among DMAC cases than among controls (OR = 0.46, p =.02). Combinations of DRB1 alleles with or without specific DMA and DMB variants showed significant differences in distributions between the cases and controls, but both of the previously associated class II alleles (DRB1*1501 and DRB1*0701) showed stronger positive associations with DMAC in the absence than in the presence of DMA*0102. Apparent joint effects of DRB1 and DM allelic combinations on occurrence and timing of DMAC suggest that class II disease relationships may be better predicted by biologically plausible interactive combinations than by polymorphisms in individual genes.     

胃肠道间质瘤的螺旋CT诊断价值

目的：探讨螺旋CT平扫和增强扫描对胃肠道间质瘤（GISTs）的诊断价值。方法：回顾性分析33例经手术、病理及免疫组化证实的GISTs患者螺旋CT表现。结果：平扫33例中，瘤体呈均匀等密度10例，肿块周边呈等密度中间略低或不均匀低密度23例。增强扫描16例中，病灶区呈中度或明显均匀强化9例，不均匀强化并可见囊状改变4例，病灶中央大片状坏死伴周边部明显强化3例。33例中良性9例，肿块直径多小于5cm，且呈圆形、类圆形，规则，边界尚清楚；恶性24例，直径多大于5cm，多数向腔外生长，边界不清楚，5例肿块中有坏死出血，4例发现转移灶。结论：螺旋CT检查对GISTs诊断虽无特异性，但可以准确定位，发现转移灶，显著提高GISTs的检出率，弥补常规胃肠道造影和内窥镜检查的不足，对GISTs术前定位和鉴别诊断均有重要价值。     

Establishment of hybridoma cell lines producing monoclonal antibodies against hepatitis B virus surface antigens (a, d, and r) and development of sensitive ELISA diagnostic test

A new treble-coated enzyme-linked immunoadsorbent assay (ELISA) kit of detecting Hepatitis B virus (HBV) surface antigen subtypes a, d and r (HBsAg-a, -d, -r) was developed by using four established hybridoma cell lines, of which two specifically secrete monoclonal antibodies (MAbs) against HBsAg-a (anti-HBsAg-a), one against -d (anti-HBsAg-d), and one against -r (anti-HBsAg-r). The approach of hybridoma cell lines' establishment were by fusing myeloma cells (SP2/0) with splenocytes from BALB/c mice immunized with a mixture of HBsAg-a, -d, -r. The ascitic MAb productivity of the four cell lines was at the titres of 1:10(6)-1:10(8). A treble-coated ELISA based HBV diagnostic kit was developed for detecting all of the three responding subtypes of HBsAgs. A 96-well ELISA microplate was coated with anti-HBSAg-a, -d, -r at a ratio of 3: 1: 0.5, with a horseradish peroxidase (HRP) conjugated anti-HBsAg-a as the labelled antibody. For clinical application, the new developed diagnostic kit detected HBsAgs of adr, adw, ayr, and ayw at a rate of lower than 0.25, 0.25, 0.5, and 0.5 ng/mL, respectively. Results indicated that this kit was more rapid and sensitive than that other current ELISA-based kits coated with a single MAb (e.g., anti-HBsAg-a).     

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ -rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ -rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ -rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA⁺ sera were tested. With δ -rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.     

Screening for cognitive impairment in older adults attending an eye clinic

PURPOSE: We conducted a cross-sectional study examining potentially modifiable factors associated with cognitive impairments (mild or severe) in older whites, African Americans and Hispanics attending an outpatient eye clinic. METHODS: In-clinic interviews and physical examinations assessed social, demographic and health information from 100 consecutive Hispanic, African-American and white adults aged > or = 55. Our primary outcome was presence of any cognitive impairment (mild or severe) using the St. Louis University Mental Status Examination (SLUMS) scale. RESULTS: Of the 100 subjects, 65 screened positive for cognitive impairments on the SLUMS cognitive instrument: 46 with mild cognitive impairment and 19 with severe impairment (possible dementia). African-American and Hispanic adults (nonwhites) were significantly more likely to have cognitive impairment compared to white adults (OR 2.80: 95% CI = 1.05-7.44), independent of age, years of education and systolic blood pressure. Subjects with diabetes also had increased odds of cognitive impairments (OR 3.28, 95% CI = 1.21-8.90) even after adjusting for relevant confounders. There was a nonsignificant trend between visual acuity impairment and cognitive impairment (p = 0.059). CONCLUSIONS: Sixty-five percent of adults aged > or = 55 attending the eye clinic screened positive for cognitive impairments, with higher rates among nonwhites and adults living with diabetes.     

Raft.1, a monoclonal antibody raised against the raft microdomain,recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line

SUMMARY: Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.