Loss of NPPA-AS1 promotes heart regeneration by stabilizing SFPQ-NONO heteromer-induced DNA repair.

The role of long non-coding RNA (lncRNA) in endogenous cardiac regeneration remains largely elusive. The mammalian cardiomyocyte is capable of regeneration for a brief period after birth. This fact allows the exploration of the roles of critical lncRNAs in the regulation of cardiac regeneration. Through a cardiac regeneration model by apical resection (AR) of the left ventricle in neonatal mice, we identified an lncRNA named natriuretic peptide A antisense RNA 1 (NPPA-AS1), which negatively regulated cardiomyocyte proliferation. In neonates, NPPA-AS1 deletion did not affect heart development, but was sufficient to prolong the postnatal window of regeneration after AR. In adult mice, NPPA-AS1 deletion improved cardiac function and reduced infarct size after myocardial infarction (MI), associated with a significant improvement in cardiomyocyte proliferation. Further analysis showed that NPPA-AS1 interacted with DNA repair-related molecule splicing factor, proline- and glutamine-rich (SFPQ). A heteromer of SFPQ and non-POU domain-containing octamer-binding protein (NONO) was required for double-strand DNA break repair, but NPPA-AS1 was competitively bound with SFPQ due to the overlapped binding sites of SFPQ and NONO. NPPA-AS1 deletion promoted the binding of SFPQ-NONO heteromer, decreased DNA damage, and activated cardiomyocyte cell cycle re-entry. Together, loss of NPPA-AS1 promoted cardiomyocyte proliferation by stabilizing SFPQ-NONO heteromer-induced DNA repair and exerted a therapeutic effect against MI in adult mice. Consequently, NPPA-AS1 may be a novel target for stimulating cardiac regeneration to treat MI.

Epigenetic control of natriuretic peptides: implications for health and disease.

The natriuretic peptides (NPs) family, including a class of hormones and their receptors, is largely known for its beneficial effects within the cardiovascular system to preserve regular functions and health. The concentration level of each component of the family is of crucial importance to guarantee a proper control of both systemic and local cardiovascular functions. A fine equilibrium between gene expression, protein secretion and clearance is needed to achieve the final optimal level of NPs. To this aim, the regulation of gene expression and translation plays a key role. In this regard, we know the existence of fine regulatory mechanisms, the so-called epigenetic mechanisms, which target many genes at either the promoter or the 3'UTR region to inhibit or activate their expression. The gene encoding ANP (NPPA) is regulated by histone modifications, DNA methylation, distinct microRNAs and a natural antisense transcript (NPPA-AS1) with consequent implications for both health and disease conditions. Notably, ANP modulates microRNAs on its own. Histone modifications of BNP gene (NPPB) are associated with several cardiomyopathies. The proBNP processing is regulated by miR30-GALNT1/2 axis. Among other components of the NPs family, CORIN, NPRA, NPRC and NEP may undergo epigenetic regulation. A better understanding of the epigenetic control of the NPs family will allow to gain more insights on the pathological basis of common cardiovascular diseases and to identify novel therapeutic targets. The present review article aims to discuss the major achievements obtained so far with studies on the epigenetic modulation of the NPs family.

Assessing cardiovascular toxicity in zebrafish embryos exposed to copper nanoparticles.

The toxicity of copper nanoparticles (CuNPs) to aquatic animals, particularly their effects on the cardiovascular system, has not been thoroughly investigated. In the present study, zebrafish embryos were used as a model to address this issue. After exposure to different concentrations (0.01, 0.1, 1, and 3 mg/L) of CuNPs for 96 h (4 to 100 h post-fertilization), cardiac parameters of the heart rate (HR), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), ejection fraction (EF), and cardiac output (CO), and vascular parameters of the aortic blood flow velocity (ABFV) and aortic diameter (AD) were examined by a video-microscopic method. Morphologically, CuNPs induced concentration-dependent pericardial edema. Although CuNPs did not alter the HR, they significantly reduced the EDV, SV, and CO at ≥0.1 mg/L, the ESV and EF at 3 mg/L, the ABFV at ≥0.1 mg/L, and the AD at ≥1 mg/L. Transcript levels of several cardiac genes, nppa, nppb, vmhc, and gata4, were also examined. CuNPs significantly suppressed nppa and nppb at ≥0.1 mg/L, gata4 at ≥0.01 mg/L, and vmhc at 1 mg/L. This study demonstrated that CuNPs can induce cardiovascular toxicity at environmentally relevant concentrations during fish embryonic development and highlight the potential ecotoxicity of CuNPs to aquatic animals.

Underlying mechanism of atrial fibrillation-associated Nppa-I137T mutation and cardiac effect of potential drug therapy.

BACKGROUND: More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved. OBJECTIVE: This study aimed to investigate the mechanism and potential treatment of NPPA mutation-associated AF. METHODS: Nppa knock-in (KI, p.I137T) rats were generated, and cardiac function was evaluated. Blood pressure was recorded using a tail-cuff system. The expression levels were measured using real-time polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot analysis, and RNA-sequence analysis. Programmed electrical stimulation, patch clamp, and multielectrode array were used to record the electrophysical characteristics. RESULTS: Mutant rats displayed downregulated expression of atrial natriuretic peptide but elevated blood pressure and enlarged left atrial end-diastolic diameter. Further, gene topology analysis suggested that the majority of differently expressed genes in Nppa KI rats were related to inflammation, electrical remodeling, and structural remodeling. The expression levels of C-C chemokine ligand 5 and galectin-3 involved in remodeling were higher, while there were declined levels of Nav1.5, Cav1.2, and connexin 40. AF was more easily induced in KI rats. Electrical remodeling included abbreviated action potentials, effective refractory period, increased late sodium current, and reduced calcium current, giving rise to conduction abnormalities. These electrophysiological changes could be reversed by the late sodium current blocker ranolazine and the Nav1.8 blocker A-803467. CONCLUSION: Our findings suggest that structural remodeling related to inflammation and fibrosis and electrical remodeling involved in late sodium current underly the major effects of the Nppa (p.I137T) variant to induce AF, which can be attenuated by the late sodium current blocker and Nav1.8 blocker.

RICH1 is a novel key suppressor of isoproterenol­ or angiotensin II­induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is one of the key processes in the development of heart failure. Notably, small GTPases and GTPase­activating proteins (GAPs) serve essential roles in cardiac hypertrophy. RhoGAP interacting with CIP4 homologs protein 1 (RICH1) is a RhoGAP that can regulate Cdc42/Rac1 and F­actin dynamics. RICH1 is involved in cell proliferation and adhesion; however, to the best of our knowledge, its role in cardiac hypertrophy remains unknown. In the present study, the role of RICH1 in cardiomyocyte hypertrophy was assessed. Cell viability was analyzed using the Cell Counting Kit­8 assay and cells surface area (CSA) was determined by cell fluorescence staining. Reverse transcription­quantitative PCR and western blotting were used to assess the mRNA expression levels of hypertrophic marker genes, such as Nppa, Nppb and Myh7, and the protein expression levels of RICH1, respectively. RICH1 was shown to be downregulated in isoproterenol (ISO)­ or angiotensin II (Ang II)­treated H9c2 cells. Notably, overexpression of RICH1 attenuated the upregulation of hypertrophy­related markers, such as Nppa, Nppb and Myh7, and the enlargement of CSA induced by ISO and Ang II. By contrast, the knockdown of RICH1 exacerbated these effects. These findings suggested that RICH1 may be a novel suppressor of ISO­ or Ang II­induced cardiomyocyte hypertrophy. The results of the present study will be beneficial to further studies assessing the role of RICH1 and its downstream molecules in inhibiting cardiac hypertrophy.

Rapid synthesis of new DNMT inhibitors derivatives of procainamide.

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.

ASA Status, NPPA/NPPB Haplotype and Coronary Artery Disease Have an Impact on BNP/NT-proBNP Plasma Levels.

Plasma concentrations of natriuretic peptides (NP) contribute to risk stratification and management of patients undergoing non-cardiac surgery. However, genetically determined variability in the levels of these biomarkers has been described previously. In the perioperative setting, genetic contribution to NP plasma level variability has not yet been determined. A cohort of 427 patients presenting for non-cardiac surgery was genotyped for single-nucleotide polymorphisms (SNPs) from the NPPA/NPPB locus. Haplotype population frequencies were estimated and adjusted haplotype trait associations for brain natriuretic peptide (BNP) and amino-terminal pro natriuretic peptide (NT-proBNP) were calculated. Five SNPs were included in the analysis. Compared to the reference haplotype TATAT (rs198358, rs5068, rs632793, rs198389, rs6676300), haplotype CACGC, with an estimated frequency of 4%, showed elevated BNP and NT-proBNP plasma concentrations by 44% and 94%, respectively. Haplotype CGCGC, with an estimated frequency of 9%, lowered NT-proBNP concentrations by 28%. ASA classification status III and IV, as well as coronary artery disease, were the strongest predictors of increased NP plasma levels. Inclusion of genetic information might improve perioperative risk stratification of patients based on adjusted thresholds of NP plasma levels.

NPPA Promoter Hypomethylation Predicts Central Obesity Development: A Prospective Longitudinal Study in Chinese Adults.

INTRODUCTION: Atrial natriuretic peptide plays a potential role in obesity with unclear molecular mechanisms. The objective of this study was to examine the association between its coding gene (natriuretic peptide A [NPPA]) methylation and obesity. METHODS: Peripheral blood DNA methylation of NPPA promoter was quantified at baseline by targeted bisulfite sequencing for 2,497 community members (mean aged 53 years, 38% men) in the Gusu cohort. Obesity was repeatedly assessed by body mass index (BMI) and waist circumference (WC) at baseline and follow-up examinations. The cross-sectional, longitudinal, and prospective associations between NPPA promoter methylation and obesity were examined. RESULTS: Of the 9 CpG loci assayed, DNA methylation levels at 6 CpGs were significantly lower in participants with central obesity than those without (all p < 0.05 for permutation test). These CpG methylation levels at baseline were also inversely associated with dynamic changes in BMI or WC during follow-up (all p < 0.05 for permutation test). After an average 4 years of follow-up, hypermethylation at the 6 CpGs (CpG2 located at Chr1:11908348, CpG3 located at Chr1:11908299, CpG4 located at Chr1:11908200, CpG5 located at Chr1:11908182, CpG6 located at Chr1:11908178, and CpG8 located at Chr1:11908165) was significantly associated with a lower risk of incident central obesity (all p < 0.05 for permutation test). CONCLUSIONS: Hypomethylation at NPPA promoter was associated with increased future risk of central obesity in Chinese adults. Aberrant DNA methylation of the NPPA gene may participate in the mechanisms of central obesity.

Serum Atrial Natriuretic Peptide, NPPA Promoter Methylation, and Cardiovascular Disease: A 10-year Follow-Up Study in Chinese Adults.

Background: Atrial natriuretic peptide (ANP) has been associated with cardiovascular disease (CVD) and related risk factors, but the clinical application is limited and the underlying mechanisms are not very clear. Here, we aimed to examine whether proANP and its coding gene methylation were associated with CVD in the Chinese population. Methods: Serum proANP and peripheral blood DNA methylation of natriuretic peptide A gene (NPPA) promoter was quantified at baseline for 2,498 community members (mean aged 53 years, 38% men) in the Gusu cohort. CVD events were obtained during 10 years of follow-up. A competing-risks survival regression model was applied to examine the prospective associations of proANP and NPPA promoter methylation with incident CVD. Results: During follow-up, 210 participants developed CVD events, 50 participants died from non-cardiovascular causes, and 214 participants were lost. Per 1-nmol/L increment of serum proANP was associated with a 22% (HR = 1.22, 95%CI: 1.03-1.44, P = 0.025) higher risk of CVD during follow-up. Of the 9 CpG sites assayed, per 2-fold increment of DNA methylation at CpG3 (located at Chr1:11908299) was significantly associated with a half lower risk of CVD (HR = 0.50, 95%CI: 0.30-0.82, P = 0.006). The gene-based analysis found that DNA methylation of the 9 CpGs at NPPA promoter as a whole was significantly associated with incident CVD (P < 0.05). Conclusions: Increased proANP and hypomethylation at NPPA promoter at baseline predicted an increased future risk of CVD in Chinese adults. Aberrant DNA methylation of the NPPA gene may participate in the mechanisms of CVD.

Identification of novel DNA methylation inhibitors via a two-component reporter gene system.

BACKGROUND: Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization. METHODS: We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives. RESULTS: A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform. CONCLUSIONS: Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.

Disruption of positive selection of thymocytes causes autoimmunity.

To differentiate into T cells, immature thymocytes must engage, through their antigen-specific T-cell receptor, peptides derived from self proteins presented by cortical epithelial cells in the thymus, a process called positive selection. Despite this requirement for self-recognition during development, mature T cells do not normally show autoreactivity. Mice injected in the thymus with procainamide-hydroxylamine, a metabolite of procainamide, develop autoimmune features resembling drug-induced lupus. Here, we show that when thymocytes undergo positive selection in the presence of procainamide-hydroxylamine, they fail to establish unresponsiveness to low affinity selecting self antigens, resulting in systemic autoimmunity.

Transformation of lupus-inducing drugs to cytotoxic products by activated neutrophils.

Drug-induced lupus is a serious side effect of certain medications, but the chemical features that confer this property and the underlying pathogenesis are puzzling. Prototypes of all six therapeutic classes of lupus-inducing drugs were highly cytotoxic only in the presence of activated neutrophils. Removal of extracellular hydrogen peroxide before, but not after, exposure of the drug to activated neutrophils prevented cytotoxicity. Neutrophil-dependent cytotoxicity required the enzymatic action of myeloperoxidase, resulting in the chemical transformation of the drug to a reactive product. The capacity of drugs to serve as myeloperoxidase substrates in vitro was associated with the ability to induce lupus in vivo.

Autoimmunity caused by disruption of central T cell tolerance. A murine model of drug-induced lupus.

A side effect of therapy with procainamide and numerous other medications is a lupus-like syndrome characterized by autoantibodies directed against denatured DNA and the (H2A-H2B)-DNA subunit of chromatin. We tested the possibility that an effect of lupus-inducing drugs on central T cell tolerance underlies these phenomena. Two intrathymic injections of procainamide-hydroxylamine (PAHA), a reactive metabolite of procainamide, resulted in prompt production of IgM antidenatured DNA antibodies in C57BL/6xDBA/2 F1 mice. Subsequently, IgG antichromatin antibodies began to appear in the serum 3 wk after the second injection and were sustained for several months. Specificity, inhibition and blocking studies demonstrated that the PAHA-induced antibodies showed remarkable specificity to the (H2A-H2B)-DNA complex. No evidence for polyclonal B cell activation could be detected based on enumeration of Ig-secreting B cells and serum Ig levels, suggesting that a clonally restricted autoimmune response was induced by intrathymic PAHA. The IgG isotype of the antichromatin antibodies indicated involvement of T cell help, and proliferative responses of splenocytes to oligonucleosomes increased up to 100-fold. As little as 5 microM PAHA led to a 10-fold T cell proliferative response to chromatin in short term organ culture of neonatal thymi. We suggest that PAHA interferes with self-tolerance mechanisms accompanying T cell maturation in the thymus, resulting in the emergence of chromatin-reactive T cells followed by humoral autoimmunity.

Metabolism of procainamide to the cytotoxic hydroxylamine by neutrophils activated in vitro.

An almost universal side effect of long-term therapy with procainamide is the appearance of serum autoantibodies and less frequently a syndrome resembling lupus erythematosus. Previous studies demonstrated that procainamide-hydroxylamine (PAHA), a metabolite generated by hepatic mixed function oxidases, was highly toxic to dividing cells, but evidence that PAHA could be formed in the circulation was lacking. This study examines the capacity of neutrophils to metabolize procainamide to reactive forms. Neutrophils activated with opsonized zymosan were cytotoxic only if procainamide was present, whereas N-acetyl procainamide, which does not induce autoimmunity, was inert in this bioassay. PAHA was detected by HPLC in the extracellular medium if ascorbic acid was present. Generation of PAHA and cytotoxic procainamide metabolites was inhibited by NaN3 and catalase but not by superoxide dismutase, indicating that H2O2 and myeloperoxidase were involved. Nonactivated neutrophils and neutrophils from patients with chronic granulomatous disease did not generate cytotoxic PAHA, demonstrating that H2O2 was derived from the respiratory burst accompanying neutrophil activation. These conclusions were supported by results of a cell-free system in which neutrophils were replaced by myeloperoxidase and H2O2 or an H2O2 generating system. These studies demonstrate the capacity of neutrophils to mediate metabolism of procainamide and establish the role of myeloperoxidase released during degranulation and H2O2 derived from the respiratory burst in the direct cooxidation of procainamide to PAHA. The profound biologic activity of this metabolite and its possible generation within lymphoid compartments implicate this process in the induction of autoimmunity by procainamide.

Torsade de pointes induced by N-acetylprocainamide.

N-Acetylprocainamide (NAPA), a class III antiarrhythmic drug, caused torsade de pointes in a 72 year old woman who had this arrhythmia on two previous occasions while being treated with quinidine and disopyramide. Initial evaluation with an intravenous infusion of NAPA indicated a favorable antiarrhythmic response. The QTC interval was prolonged, but the 2.4 ms/microgram per ml incremental QTC interval lengthening caused by NAPA was not greater than usual. During subsequent oral therapy with NAPA, torsade de pointes developed at plasma levels of this drug that appeared to be well tolerated during the initial evaluation.

Effects of N-acetylprocainamide on experimental atrial flutter and atrial electrophysiologic properties in conscious dogs with sterile pericarditis: comparison with the effects of quinidine.

N-acetylprocainamide (NAPA) is said to have class III antiarrhythmic drug properties. The effects of NAPA (25 mg/kg intravenously) on sustained, stable, reentrant atrial flutter induced in 12 conscious dogs using a sterile pericarditis model were studied and compared with the effects of quinidine (5 mg/kg intravenously) given on a different day in 10 of the same 12 dogs. The effects of these drugs on atrial excitability, the atrial effective refractory period and intraatrial conduction time measured during rapid atrial pacing performed during sinus rhythm were also compared. The mean NAPA and quinidine serum levels were 17.7 and 7.1 micrograms/ml, respectively. Both NAPA and quinidine immediately prolonged the atrial flutter cycle length in all dogs, from 118 +/- 15 to 141 +/- 18 ms and from 119 +/- 17 to 153 +/- 21 ms, respectively (both p less than 0.001), and then terminated atrial flutter in 11 of the 12 NAPA studies and in 6 of the 10 quinidine studies. Neither drug affected atrial excitability. Both NAPA and quinidine increased the atrial effective refractory period significantly, from 138 +/- 17 to 168 +/- 20 ms (p less than 0.001) and from 136 +/- 14 to 148 +/- 16 ms (p less than 0.01), respectively. NAPA did not change intraatrial conduction time measured during atrial pacing at 150 beats/min, but during atrial pacing at 300 beats/min, it prolonged it from 51 +/- 9 to 54 +/- 10 ms (p less than 0.05), and at 400 beats/min, from 52 +/- 10 to 64 +/- 13 ms (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic effects of intravenous sematilide in patients with congestive heart failure: a class III antiarrhythmic agent without cardiodepressant effects.

OBJECTIVES: This study sought to evaluate the hemodynamic effects of intravenous sematilide hydrochloride, a selective class III antiarrhythmic agent, in patients with heart failure and left ventricular systolic dysfunction. BACKGROUND: Class I antiarrhythmic agents, which primarily slow conduction, can depress ventricular function, particularly in patients with heart failure. In contrast, pure class III agents, which selectively prolong repolarization, do not adversely affect hemodynamic variables in animal models, but there are no data evaluating their hemodynamic effects in humans. METHODS: In 39 patients with congestive heart failure and a left ventricular ejection fraction < 40%, hemodynamic and electrocardiographic measurements were obtained at baseline, after a loading dose and during a maintenance infusion of intravenous sematilide using either a low (0.75 then 0.3 mg/min) or high dose (1.5 then 0.6 mg/min) regimen. The study had an 80% power to detect clinically meaningful differences in hemodynamic variables. RESULTS: Both low (n = 20) and high (n = 19) dose sematilide infusions produced dose-dependent increases in QT interval (5 +/- 8% [mean +/- SD] and 18 +/- 10%, respectively) and corrected QT interval (4 +/- 8% and 14 +/- 10%), and high dose sematilide decreased heart rate by 7 +/- 10% (all p < 0.025 vs. baseline). Neither dose regimen had a statistically significant effect on any other hemodynamic variable, including mean arterial, right atrial, pulmonary artery and pulmonary capillary wedge pressures; cardiac index, stroke volume, systemic and pulmonary vascular resistances; and left ventricular stroke work index. Sematilide showed no adverse hemodynamic effects in patients with left ventricular ejection fraction < or = 25% or > 25% and in patients with cardiac index < 2 or > or = 2 liters/min per m2. Sustained polymorphic ventricular tachycardia (n = 1) and excessive QT prolongation (n = 4) were seen during the high dose. CONCLUSIONS: Sematilide, in the doses administered, prolonged repolarization but did not alter hemodynamic variables in patients with heart failure. These data suggest that class III antiarrhythmic agents, which selectively prolong repolarization, are not cardiodepressant but may be proarrhythmic in humans, especially at high doses.

A nondeletional mechanism for central T-cell tolerance.

To be positively selected, immature thymocytes must receive signaling through their T-cell receptor (TCR), and engagement of relatively low-affinity self-peptides permits further T-cell maturation. However, mature T cells no longer overtly respond to such low-affinity antigens, indicating that T cells acquire a higher threshold for activation during thymopoiesis. We wondered whether partial interference in positive selection could produce T cells that respond to the selecting self-peptide. This possibility was tested by injecting procainamide-hydroxylamine (PAHA), a lupus-inducing drug, into the thymus of adult normal mice. Three weeks after the second injection, IgG antichromatin antibodies appeared in the circulation and remained for several months. The murine antichromatin antibodies reacted with the (H2A-H2B)-DNA subnucleosome complex, the predominant specificity in patients with procainamide-induced lupus. In thymus organ and reaggregate cultures, PAHA had no effect on negative selection of T cells with high affinity for a co-present antigen, but acted on CD4+ CD8+ immature T cells as they underwent positive selection. TCR transgenic T cells specific to cytochrome c peptide 88-104 acquired the capacity to respond to the low-affinity analogue at position 99 (lys-->ala) if PAHA was present during their development. PAHA also blocked the capacity of a T-cell line to become anergic after anti-CD3 treatment, suggesting that PAHA prevents the production of negative regulators that accumulate in response to partial signaling through the TCR. These results are consistent with the view that T cells acquire self-tolerance during positive selection, and disruption of this process can result in autoreactive T cells and systemic autoimmunity.

Trimethoprim inhibition of the renal clearance of procainamide and N-acetylprocainamide.

To test the effect of trimethoprim (an antibiotic commonly administered with sulfamethoxazole) on the disposition of the antiarrhythmic procainamide hydrochloride and its active metabolite N-acetylprocainamide, 10 healthy men received 1 g of procainamide hydrochloride orally on two occasions, coadministered with placebo or trimethoprim (100 mg twice a day for 2 days before and then 200 mg with the procainamide dose). Trimethoprim decreased the mean (+/- SD) renal clearance by 45% after the dose of procainamide was administered (487 +/- 129 vs 267 +/- 123 mL/min) and that of N-acetylprocainamide by 26% (275 +/- 78 vs 192 +/- 82 mL/min) compared with placebo. The mean area under plasma concentration--time curve 0 to 12 hours after dosing increased 39% for procainamide (19.8 +/- 4.8 vs 27.6 +/- 7.2 mg.h/L) and 27% for N-acetylprocainamide (9.1 +/- 2.1 vs 11.4 +/- 2.8 mg.h/L). The corrected QT electrocardiographic interval at 2 hours after the procainamide dose was 0.40 +/- 0.02 second with placebo and 0.43 +/- 0.03 second with trimethoprim. Trimethoprim may increase procainamide and N-acetylprocainamide plasma concentrations, resulting in increased pharmacodynamic response apparently caused by the competition for renal tubular cationic secretion.

N-substituted benzamides inhibit nuclear factor-kappaB and nuclear factor of activated T cells activity while inducing activator protein 1 activity in T lymphocytes.

N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.