Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody

Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.     

Clinical results of drug eluting stents compared to bare metal stents for patients with ST elevation acute myocardial infarction

OBJECTIVE: To investigate the clinical outcomes in patients with ST segment elevation acute myocardial infarction (STEMI) treated with drug eluting stents (DES) versus a matched control group of patients with STEMI treated with bare metal stents (BMS). METHODS: This registry included 122 patients with STEMI undergoing primary coronary angioplasty with DES implantation at our institution. The control group consisted of 506 patients implanted with BMS, who were matched for age, infarct location, and diabetic status. The incidences of major adverse cardiac events (MACE) including target vessel/lesion revascularization (TVR/TLR) and stent thrombosis were assessed up to 12 months. RESULTS: Twelve months follow up showed a non-significant trend towards reduced deaths (3.3% versus 7.1%, P=0.1), significantly reduced recurrent MI (0.0% versus 6.1%, P=0.02), TVR (5.7% versus 15.2%, P=0.006) and TLR (2.5% versus 14.0%, P=0.004) events in the DES group as compared to BMS group. The composite incidences of MACE at 12 months follow-up was lower in the DES group (11.5%) as compared to the BMS group (21.3%, P=0.01). CONCLUSION: According to our experiences, the use of DES in STEMI is safe and effective as compared to BMS. DES was effective in reducing the incidence of restenosis outcomes and overall adverse cardiac events up to 12 months.     

Treatment of Induced Murine SLE with a Peptide Based on the CDR3 of an Anti-DNA Antibody Reverses the Pattern of Pathogenic Cytokines

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA monoclonal antibody that bears the 16/6 idiotype (Id) was shown previously to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated responses. When injected, concomitant with active immunization with the pathogenic human anti-DNA, 16/6 Id + mAb, pCDR3 inhibited the proliferation of LN-derived T cells stimulated in vitro with the 16/6 Id mAb. The inhibition of the specific proliferative responses could be reversed by the addition of exogenous IL-2 to the cultures. Analysis of secreted cytokine profile in supernatants of these cultures demonstrated that pCDR3 treatment reduced significantly the levels of both IL-2 and IFN- &#110 that were elevated further in cells of the 16/6 Id-immunized mice. The CDR3-based peptide was shown here to immunomodulate in vivo experimental SLE, induced by the human anti-DNA 16/6 Id + antibody. The beneficial effects of pCDR3 on the clinical manifestations of SLE were associated with downregulation of the Th1-type (IL-2, IFN- &#110 ) and proinflammatory (TNF- &#102 ) cytokines, whereas the immunosuppressive cytokine TGF- &#103 was up regulated.     

Effect of clopidogrel pretreatment on angiographic and clinical outcomes in patients undergoing primary percutaneous coronary intervention for ST-elevation acute myocardial infarction

Pretreatment with clopidogrel before elective primary percutaneous coronary intervention (PCI) has been shown to reduce ischemic complications. There are limited data about the value of clopidogrel pretreatment in the setting of PCI for ST-elevation myocardial infarction (STEMI). We aimed to examine the effect of clopidogrel preloading on angiographic and clinical outcomes in patients with STEMI who were treated with PCI. We conducted a prospective registry of all patients treated with primary PCI for STEMI from March 2003 to June 2006. Excluded were patients with cardiogenic shock. For the current analysis, patients (n = 292) were allocated into 2 groups. One group received clopidogrel loading dose before PCI (in the emergency department or coronary care unit, n = 165); the other,immediately after PCI (n = 127). TIMI myocardial perfusion (TMP) grade at the end of PCI and 30-day and 6-month clinical outcomes were assessed. Clinical characteristics were similar among the groups. However, patients pretreated with clopidogrel were more likely to receive aspirin and beta blockers before the current admission. TMP grade 3 occurred in a higher proportion of patients in the clopidogrel pretreatment group than in the no-pretreatment group (85% vs 71%, p = 0.01). Multivariate logistic regression analysis showed that clopidogrel pretreatment was associated with an odds ratio of 2.2 for TMP grade 3 (1.2 to 3.9, p = 0.01). Furthermore, the incidence of reinfarction at 30 days was lower in the pretreatment group (0% vs 3.2%, respectively, p = 0.04). In conclusion, these findings support the early use of clopidogrel in patients with STEMI who are treated with primary PCI.     

Effect of Different Surface Treatments of Lithium Disilicate on the Adhesive Properties of Resin Cements

The aim of the current study was to evaluate the influence of hydrofluoric (HF) acid concentration and conditioning time on the shear bond strength (SBS) of dual cure resin cement to pressed lithium disilicate ceramic compared to treatment with an Etch and Prime self-etching glass-ceramic primer (EP). A total of 100 samples of pressed lithium disilicate (IPS e.max Press, Ivoclar Vivadent) were randomly divided into five groups (n = 20) according to surface treatment: two different concentrations of HF (5% or 9%), for different durations (20 or 90 s), or treatment with EP. Adhesion of light-cured resin cement to the treated surface was tested by the SBS test. The substrate surfaces of the specimen after failures were examined by SEM. Data were analyzed using Weibull distribution. The highest cumulative failure probability of 63.2% of the shear bond strength (η parameter) values was in the 9% HF −90 s group (17.71 MPa), while the lowest values were observed in the 5% HF −20 s group (7.94 MPa). SBS values were not affected significantly by the conditioning time (20 s or 90 s). However, compared to treatment with 5% HF, surface treatment with 9% HF showed a significantly higher η (MPa) as well as β (reliability parameter). Moreover, while compared to 9% HF for 20 s, EP treatment did not differ significantly in SBS values. Examination of the failure mode revealed a mixed mode of failure in all the groups. Within the limits of this study, it is possible to assume that IPS e.max Press surface treatment with 9% HF acid for only 20 s will provide a better bonding strength with resin cement than using 5% HF acid.     

Effect of enalapril and nifedipine on orthostatic hypotension in older hypertensive patients

OBJECTIVE: To compare the effect of enalapril with long-acting nifedipine on orthostatic hypotension in older patients. DESIGN: A prospective, double blinded, cross-over study. SETTING: The outpatient clinic of a university hospital. PARTICIPANTS: Thirty-nine patients aged 65 years or older with systolic blood pressure (SBP) of 140-190 mm Hg and diastolic blood pressure (DBP) of 90-110 mm Hg. INTERVENTION: Enalapril 5-20 mg od or nifedipine 30-90 mg od for 8 weeks, followed by 4 weeks washout and cross-over for a second 8-week period. MEASUREMENTS: Supine and standing 0-, 1-, and 5-minutes blood pressure was recorded before and at the end of each treatment period. RESULTS: At baseline, SBP was 158.8 +/- 8.7 mm Hg, and DBP was 97.1 +/- 5.9 mm Hg. There was a decline in SBP of 6.1 +/- 2.7 mm Hg and 8.4 +/- 4.1 mm Hg after 1 and 5 minutes of standing, respectively. Both agents caused a significant decline in supine blood pressure. Enalapril: supine SBP 158.8 +/- 8.7 to 143 +/- 7.3 mm Hg; supine DBP 97.1 +/- 5.9 to 85.1 +/- 5.1 mm Hg (P = .0001). The drop in SBP after standing for 5 minutes was only 2.4 +/- 1.6 mm Hg with no change in diastolic values. A > or = 10 mm Hg drop in SBP was observed in only three patients, and no patient experienced a decline of 20 mm Hg or more. Nifedipine: supine SBP: 160.3 +/- 9 to 145.3 +/- 8.1 mm Hg; supine DBP: 96.3 +/- 5.7 to 86.3 +/- 5.8 (P = .0001). Nifedipine induced an orthostatic decline in SBP values; there was an 8.7 +/- 4.8 mm Hg difference between supine and 5 minutes standing values (P = .0005) without change in diastolic values. An orthostatic decline in SBP of > or = 10 mm Hg occurred in 13 patients, and there was a drop of > or = 20 mm Hg in six patients. The cross-over of enalapril and nifedipine reproduced the hypotensive effect and reversed the postural effect. (P = .0002 nifedipine vs enalapril) CONCLUSIONS: Enalapril and nifedipine were equipotent in reducing supine blood pressure levels. Enalapril also reduced the number of orthostatic episodes significantly, whereas nifedipine aggravated this phenomenon.     

Effects of Aging Torque Controllers on Screw Tightening Force and Bacterial Micro-Leakage on the Implant-Abutment Complex

Aim: We assess the accuracy of torque controllers after several aging processes and the bacterial leakage on implant-abutment complexes (IAC). Methods: A total of 12 spring-type and 12 friction-type torque controllers and 48 IAC (24 conical and 24 hexagonal connections) were evaluated. Chemical, mechanical, temperature, and pressure-aging methods were applied individually to replicate clinical use. Torque controller accuracy was analyzed before and after aging using a calibrated gauge. To assess bacterial leakage, the IAC were suspended in a bacterial medium for 24 h. Direct Contact Test (DCT) and Polymerase Chain Reaction Test (RT-PCR) analyzed the infiltration of F. nucleatum and P. gingivalis into the IAC micro-gap. Results: A significant decrease in torque after 10 days of aging was found. The spring-type torque controller was affected the most, regardless of the aging method (P < 0.05). PCR results indicated that all groups exhibited significantly more bacterial leakage, regardless of the method used (P < 0.05). The conical IAC demonstrated more bacterial leakage of P. gingivalis compared with the hexagonal IAC (P = 0.07). DCT found bacterial growth in the IAC only before aging and was not identified after aging. Conclusion: Aging affects torque accuracy. A reduction in force was noticed after 10 days. The conical IAC exhibits more bacterial leakage, although this was not statistically significant.     

Genomic context sensitivity of insulator function

The specificity of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which can act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But not all CTCF sites genome-wide function as insulator elements, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position reveals distinct expression patterns subject to genomic context, including a compartment of enhancer blocker reporter integrations with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles and their decomposition into sensitivity to both local and distant DNase I hypersensitive sites. Furthermore, using a single-cell expression approach to test the effect of integrated reporters for differential expression of nearby endogenous genes reveals that CTCF insulator elements do not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight into genomic regulatory compartmentalization and its influence on the determinants of promoter–enhancer specificity.

A boundary model offers an attractive paradigm for understanding regulatory specificity in mammalian genomes through the delineation of independent regulatory domains. Insulators are a class of genomic regulatory elements that block interaction of enhancers with their cognate promoters (Phillips and Corces 2009). Enhancer blocker activity is canonically defined by a reporter assay that interposes a candidate insulator element between a weak promoter and an enhancer (Chung et al. 1993), while barrier insulators protect transgenes from silencing owing to spreading of heterochromatin (West et al. 2002). Insulators have also been used to counter genotoxicity from transgene enhancer activation of endogenous oncogenes (Li et al. 2009; Liu et al. 2015). Known insulators such as the chicken beta-globin hypersensitive site 4 element or the Igf2/H19 imprinting control region (Bell and Felsenfeld 2000) are composite elements with enhancer blocker, barrier, and other activities (Dickson et al. 2010), and often have secondary functions, such as silencers (Qi et al. 2015).The architectural protein CTCF is the only known vertebrate insulator protein, and its binding can confer a potent enhancer blocking effect (Phillips and Corces 2009). Additionally, binding sites for CTCF colocalize with genomic features such as topologically associated domain boundaries (Dixon et al. 2012), but direct functional analysis of these sites is impeded by the difficulty of genome engineering at the relevant scales. Although binding affinity, DNA methylation, and recognition sequence orientation appear to confer some specificity for CTCF sites involved in domain organization (de Wit et al. 2015; Guo et al. 2015; Sanborn et al. 2015), these factors alone remain inadequate to distinguish true insulator elements impacting expression of nearby genes from the ∼100,000 CTCF sites genome-wide (Maurano et al. 2015; Tycko et al. 2019). Stably integrated reporter assays have shed light on the mechanics of insulator function, but such methods do not assess interaction with the surrounding endogenous genomic elements (Walters et al. 1999). In contrast, integrated barcoded reporter assays (Akhtar et al. 2013; Maricque et al. 2019; Moudgil et al. 2020) offer the potential to directly assess the interaction between novel CTCF sites and the endogenous genomic landscape.Here, we aim to functionally characterize endogenous genomic regulatory elements through their effect on integrated reporters. We describe a high-throughput randomly integrated barcoded reporter platform based on a previously described enhancer blocker construct interposing a potent CTCF insulator element (Liu et al. 2015) between a weak promoter and a potent enhancer. We integrate these reporters, both with and without insulator elements, randomly throughout the genome of K562 erythroleukemia cells using the Sleeping Beauty transposase system. We use the unique reporter barcodes to map individual insertion locations and enable position-specific readout of genomic context effects. Finally, we apply single-cell RNA-seq (scRNA-seq) to detect specific reporter integrations that perturb endogenous gene expression.     

Werner syndrome protein: biochemical properties and functional interactions

Werner syndrome is a premature aging syndrome displaying numerous signs and symptoms found in normal aging. The disease is associated with a mutation in the WRN gene. We have purified the Werner protein (WRN) and studied its biochemical activities and its protein interactions. WRN is a helicase and an exonuclease and also has an associated ATPase activity. WRN interacts physically and functionally with replication protein A (RPA), which stimulates its helicase activity. We have studied the WRN exonuclease activity and found that it can be blocked by certain DNA lesions and not by others. Thus, while WRN does not bind to DNA damage, it may have properties that allow it to sense the presence of damage in DNA. More recently we have found other protein interactions that involve physical and functional interactions with WRN, which could suggest a role for WRN in DNA repair.     

Repair of pig dura in vivo using temperature controlled CO(2) laser soldering

BACKGROUND AND OBJECTIVES: The purpose of this study was to demonstrate that laser soldering might be successfully used for closing holes or cuts in the dura layer, which encapsulates the brain. STUDY DESIGN/MATERIALS AND METHODS: A temperature controlled fiberoptic CO(2) laser system and albumin solder were used for spot soldering of fascia patches to holes in the dura of farm pigs, in vitro and in vivo. RESULTS: The mean burst pressure of the soldered patches in the in vitro experiments was 190 +/- 88 mm Hg-significantly higher than typical maximum CSF pressure of 15 mm Hg. In the in vivo experiments the pigs showed no postoperative complications. Histopathological studies exhibited an accepted level of inflammatory reaction and showed no thermal damage to the underlying brain tissue. CONCLUSIONS: It has been clearly demonstrated that temperature controlled laser soldering is a very useful technique for the repair of the dura. It provides significant advantages over standard closure techniques: it is easy to apply, the bond is strong and watertight and the procedure is likely to be much faster than suturing. This research work will lead to clinical trials.