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.     

Novel micro-crosslinked poly(organophosphazenes) with improved mechanical properties and controllable degradation rate as potential biodegradable matrix

As biodegradable materials, linear polyphosphazenes undergo rapid hydrolysis degradation but exhibit poor mechanical properties. Blending with biodegradable polyesters or inorganic particles strengthen their mechanical properties but give rise to slower degradation rate. To balance the mechanical properties and the degradation rate, micro-crosslinked polyphosphazenes were synthesized in this study. Their glass transition temperatures, mechanical properties, and in vitro degradation behavior were investigated. 2-hydroxyethyl methacrylate (HEMA) was firstly attached to the side chain along with glycine ethyl ester to prepare co-substituted poly(organophosphazene) with pendant ethenyl substituents. The co-substituted poly(organophosphazene) was blended with HEMA or acrylic acid (AA) followed by a free radical polymerization to prepare micro-crosslinked poly(organophosphazenes). The resulting crosslinked polymers showed two separate glass transition temperatures depending on the HEMA or AA feed. Incorporation of crosslinking affected the mechanical properties positively. Crosslinked poly(organophosphazenes) showed an approximately 11-17 fold increase in terms of modulus of elasticity when compared to the linear counterpart. In vitro degradation tests indicated that HEMA-crosslinked polymers hydrolyzed at a retarded rate while AA-crosslinked polymers hydrolyzed at a moderate rate compared to linear polymers.     

Toosendanin induces outgrowth of neuronal processes and apoptosis in PC12 cells

In the present study, the effects of toosendanin on cell differentiation and apoptosis were investigated in PC12 cells. The results showed that after 24-48 h of culture in a medium containing toosendanin (approximately 1-10x10(-7) M), cell differentiation and outgrowth of neuronal processes were promoted. Combined treatment with toosendanin and a calcium channel blocker, nifedipine or omega-conotoxin GVIA, resulted in a significant inhibition of the toosendanin-induced effects. Pretreatment of PC12 cells with BAPTA-AM also inhibited the toosendanin-induced effects; however, these effects were not inhibited by pertussis toxin and H-7 in the medium. Toosendanin also induced cell apoptosis. Based on the DNA content determined by flow cytometric analysis, the number of apoptotic cells significantly increased when the incubation time in the toosendanin-containing medium was lasted up to 72 h. Toosendanin at a higher concentration (> or =1 x 10(-6) M) caused cell death while it had no effect on cell division at concentrations lower than 1 x 10(-7) M.     

Intergeneration CAG expansion and contraction in a Chinese HD family.

The prevalence of juvenile-onset Huntington's disease (HD) is about ten times lower than adult HD. Here we report a Chinese HD family showing both intergeneration CAG expansion and contraction. The expansion resulted from a paternal transmission which leads to juvenile-onset HD for a 17-year-old Chinese boy (III-5). More interestingly, a contraction was noticed in a maternal transmission (III-3), which changed the CAG repeat from an expanded, disease-causing allele (48 repeats) to a normal or intermediate allele (34 repeats). Of note, the contraction resulted in a deletion of 14 CAG repeats, which is much larger than previously reported contractions. Our results are consistent with previous observations in Western Caucasians that juvenile-onset HD is more likely inherited through the male germline.     

Functional evidence of decreased tumorigenicity associated with monochromosome transfer of chromosome 14 in esophageal cancer and the mapping of tumor-suppressive regions to 14q32

Despite the abundant evidence of high allelic loss of chromosome arm 14q in human cancers, tumor-suppressor genes mapped to this chromosome have yet to be identified. To narrow the search for candidate genes, we performed monochromosome transfer of chromosome 14 into an esophageal carcinoma cell line, SLMT-1 S1. Statistically significant suppression of the tumorigenic potential of microcell hybrids containing the transferred chromosome 14 provided functional evidence that tumor-suppressive regions of chromosome 14 are essential for esophageal cancer. Tumor segregants emerging in nude mice during the tumorigenicity assay were analyzed by detailed PCR-microsatellite typing to identify critical nonrandomly eliminated regions (CRs). A 680-kb CR mapped to 14q32.13 and an approximately 2.2-Mb CR mapped to 14q32.33 were delineated. Dual-color BAC FISH analysis of microcell hybrids and tumor segregants verified the selective loss of the 14q32.13 region. In contrast, similar transfers of an intact chromosome 11 into SLMT-1 S1 did not significantly suppress tumor formation. These functional complementation studies showing the correlation of tumorigenic potential with critical regions of chromosome 14 validated the importance of the 14q32 region in tumor suppression in esophageal cancer. The present study also paved the path for further identification of novel tumor-suppressor genes that are relevant to the molecular pathogenesis of esophageal cancer.     

Impact of Dissection after Drug-Coated Balloon Treatment of De Novo Coronary Lesions: Angiographic and Clinical Outcomes

PurposeDissection after plain balloon angioplasty is required to achieve adequate luminal area; however, it is associated with a high risk of vascular events. This study aimed to examine the relationship between non-flow limiting coronary dissections and subsequent lumen loss and long-term clinical outcomes following successful drug-coated balloon (DCB) treatment of de novo coronary lesions.Materials and MethodsA total of 227 patients with good distal flow (Thrombolysis in Myocardial Infarction flow grade 3) following DCB treatment were retrospectively enrolled and stratified according to the presence or absence of a non-flow limiting dissection. The primary endpoint was late lumen loss (LLL) at 6-month angiography, and the secondary endpoint was target vessel failure (TVF, a composite of cardiac death, target vessel myocardial infarction, target vessel revascularization, and target vessel thrombosis).ResultsThe cohort consisted of 95 patients with and 132 patients without a dissection. There were no between-group differences in LLL (90.8%) returning for angiography at 6 months (0.05±0.19 mm in non-dissection and 0.05±0.30 mm in dissection group, p=0.886) or in TVF (6.8% in non-dissection and 8.4% in dissection group, p=0.799) at a median follow-up of 3.4 years. In a multivariate analysis, the presence of dissection and its severity were not associated with LLL or TVF. Almost dissections (93.9%) were completely healed, and there was no newly developed dissection at 6-month angiography.ConclusionThe presence of a dissection following successful DCB treatment of a de novo coronary lesion may not be associated with an increased risk of LLL or TVF (Impact of Drug-coated Balloon Treatment in de Novo Coronary Lesion; {"type":"clinical-trial","attrs":{"text":"NCT04619277","term_id":"NCT04619277"}}NCT04619277).     

皮下通道型胆囊肝胆管成形术治疗肝胆管结石 和胆管狭窄的临床研究

目的：总结皮下通道型胆囊肝胆管成形术的临床效果。方法：统计了1994年以来我院76例行STHG手术患者的适应症、临床效果及术后早期并发症。并选择同期实施的125 列行传统胆肠吻合（CJ）的患者进行对照。结果：STHG的手术适应症与传统胆肠吻合基本相同;STHG组患者的手术时间、住院时间、术中出血量明显少于CJ组,术后恢复良好,仅2例次并发症。结论;该术式既保存了胆囊及Oddi括约肌功能,又保证了胆汁的生理流向;防止了肠液的反,所以避免了消化功能紊乱,防止了反 流性胆管炎,对患者的影响小,是一种较为理想的治疗肝胆管结石和肝门胆管狭窄的术式。