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.

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.

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.

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.

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.

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.

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.

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.

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.

Lesion evidence that two distinct regions within prefrontal cortex are critical for n-back performance in humans.

Although prefrontal cortex is clearly important in executive function, the specific processes carried out by particular regions within human prefrontal cortex remain a matter of debate. A rapidly growing corpus of functional imaging work now implicates various areas within prefrontal cortex in a wide range of "executive" tasks. Loss-of-function studies can help constrain the interpretation of such evidence by testing to what extent particular brain areas are necessary for a given cognitive process. Here we apply a component process analysis to understand prefrontal contributions to the n-back task, a widely used test of working memory, in a cohort of patients with focal prefrontal damage. We investigated letter 2-back task performance in 27 patients with focal damage to various regions within prefrontal cortex, compared to 29 demographically matched control subjects. Both "behavior-defined" approaches, using qualitative lesion analyses and voxel-based lesion-symptom mapping methods, and more conventional "lesion-defined" groupwise comparisons were undertaken to determine the relationships between specific sites of damage within prefrontal cortex and particular aspects of n-back task performance. We confirmed a critical role for left lateral prefrontal cortex in letter 2-back performance. We also identified a critical role for medial prefrontal cortex in this task: Damage to dorsal anterior cingulate cortex and adjacent dorsal fronto-medial cortex led to a pattern of impairment marked by high false alarm rates, distinct from the impairment associated with lateral prefrontal damage. These findings provide converging support for regionally specific models of human prefrontal function.

N-substituted 4-aminobenzamides (procainamide analogs): an assessment of multiple cellular effects concerning ion trapping.

Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-kappaB inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPase-dependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma). Giant vacuole formation, of confirmed trans-Golgi origin (labeled with C5-ceramide, p230, golgin-97), is a cellular response to all tested amines in the series (> or = 2.5 mM), including triethylamine. These drugs and the V-ATPase inhibitor bafilomycin A1 inhibited smooth muscle cell proliferation, suggesting that acidification of a cellular compartment is essential to cell division. The cytotoxicity was maximal with metoclopramide, and this effect was minimally influenced by bafilomycin A1; furthermore, metoclopramide (2.5 mM) induced apoptosis in tumor cells as judged by poly(ADP-ribose)polymerase (PARP) cleavage. Triethylamine and procainamide exhibit a low level of cytotoxicity variably reduced by bafilomycin co-treatment. In Morris cells, the secretion of alpha-fetoprotein is inhibited by amines, consistent with the impairment of the secretory pathway. The most highly substituted 4-aminobenzamides are significant NF-kappaB inhibitors in smooth muscle cells. Although some effects of 4-aminobenzamides are independent of V-ATPase-driven ion trapping (inhibition of NF-kappaB nuclear translocation, agent-specific cytotoxicity, PARP cleavage), other effects are dependent on this phenomenon (vacuolization, a component of the cytotoxicity, inhibition of secretion).

[Anticholinergic effect of procainamide and its sulfonylcarbamide derivatives on electromechanical activity in guinea pig atrium]. / Prokainamido ir jo sulfonilkarbamidiniu analogu anticholinerginis poveikis juru kiaulytes priesirdziu elektromechaniniam aktyvumui.

The aim of the study was to investigate the effect of class IA antiarrhythmic drug procainamide and its new derivatives PA20, PA23, PA28 and PA53 on carbachol-induced action potential duration and contraction force in guinea pig atrial muscles. Experiments were carried out using standard method of registration of myocardium electromechanical activity. Under control conditions (perfusion of atrial strips with Tyrode solution), mean action potential duration measured at 90% (AP90) and 50% (AP50) of repolarization were 92.5+/-4.5 ms and 44.4+/-2.9 ms (n=18), respectively, and contraction force was of 2.7+/-0.5 mN (n=12). Carbachol (10(-6) M), an agonist of muscarinic acetylcholine receptor and activator of K(Ach) channels, markedly decreased AP90 to 32.4+/-2.4%, AP50 - to 25.4+/-2.2% (n=18) (p<0.001) and contraction force - to 24.2+/-5.8% (n=16) (p<0.05), vs. control. Procainamide and its new sulfonylcarbamide derivatives PA20, PA23, PA28 (10(-5)-3x10(-3) M), and PA53 (10(-5)-10(-3) M) reversed the carbachol-induced action potential duration shortening at different extent: procainamide derivative PA20 (N-cyclohexylsulfonylcarbamide fragment is linked up to benzene ring) had the most potent anticholinergic effect on action potential duration and contraction force of guinea pig atrial muscles. N-ethylsulfonylcarbamide fragment in PA23 or permanent positive charge of aliphatic nitrogen in PA28 had a weaker anticholinergic effect (similar to procainamide action) on action potential duration and contraction force than PA20. The weakest anticholinergic effect was induced by procainamide derivative PA53 with isosteric form of sulfonylcarbamide.

Differential usage of IkappaBalpha and IkappaBbeta in regulation of apoptosis versus gene expression.

In this study we use the N-substituted benzamides declopramide (3-CPA) and N-acetyl declopramide (Na-3-CPA) to investigate the involvement of the transcription factor NF-kappaB in the induction of apoptosis and surface immunoglobulin kappa (Igkappa) expression in the mouse pre-B cell line 70Z/3. We first showed that 3-CPA-induced apoptosis at doses around 500 microM and that the 3-CPA-induced apoptosis could be suppressed by over-expression of the Bcl-2 protein. Na-3-CPA was shown to be non-apoptotic at doses up to 1-2 mM. On the other hand, Na-3-CPA inhibited LPS-induced Igkappa expression while 3-CPA had no effect. Further analysis showed that while 3-CPA inhibited breakdown of IkappaBalpha, Na-3-CPA inhibited breakdown of IkappaBbeta. In addition, we used a 70Z/3 cell line expressing a dominant negative IkappaBalpha (70Z/3(deltaNIkappaBalpha)). The 70Z/3(deltaNIkappaBalpha) cell line was shown to be more sensitive to apoptosis and cytotoxicity induced by 3-CPA as well as by LPS, probably due to a defect in NF-kappaB rescue mechanism. Taken together, our data implicate distinct roles for IkappaBalpha and IkappaBbeta in regulating various NF-kappaB activities.

Comparison of the in vivo electrophysiological and proarrhythmic effects of amiodarone with those of a selective class III drug, sematilide, using a canine chronic atrioventricular block model.

Amiodarone effectively blocks both the sodium and calcium channels and beta-adrenoceptors, in addition to blocking several potassium currents including IKr, IKs, Ito, IK1, IKACh and IKNa. The incidence of clinical torsade de pointes (TdP) associated with amiodarone has been reported to be low and the present study compared the proarrhythmic potential of amiodarone with that of a selective IKr channel blocker, sematilide, using a canine chronic atrioventrucular block model. Amiodarone or sematilide (3 and 30 mg/kg; n=4 for each group) was administered orally without anesthesia under continuous ECG monitoring. Both drugs prolonged the QT interval, although the onset was faster for sematilide. The high dose of sematilide induced TdP in 3 of 4 animals, which caused their death, but neither the low dose of sematilide nor the 2 dosages of amiodarone induced lethal ventricular arrhythmias. These results suggest that IKr channel inhibition by amiodarone with its additional ion channel blocking action may contribute to the prevention of TdP.

Effects of a typical I(Kr) channel blocker sematilide on the relationship between ventricular repolarization, refractoriness and onset of torsades de pointes.

The effects of a typical I(Kr) channel blocker sematilide on the relationship between ventricular repolarization, refractoriness and onset of torsades de pointes (TdP) were studied using the canine isolated, blood-perfused ventricular septum preparation with monophasic action potential (MAP) recording. Intracoronary infusion of sematilide (10-100 microg/min) prolonged the repolarization phase and effective refractory period, the extent of which was greater in the former than in the latter, resulting in prolongation of terminal repolarization process. Prolonging the basic pacing cycle length from 400 to 600 ms and/or increasing the drug doses enhanced each of these actions. Reverse use-dependence was obvious in the drug-induced prolongation of MAP duration, but it was less clear in the effective refractory period. More importantly, during sematilide infusion, in preparations paced at longer basic cycle length of 600 - 2000 ms, TdP-like polymorphic ventricular tachycardia was repeatedly induced by an extra-stimulus applied on the terminal repolarization phase, which indicates the appearance of electrically vulnerable period. Prolonging the basic pacing cycle length and/or increasing the drug doses prolonged this electrically vulnerable period in parallel with the terminal repolarization phase. These results suggest that prolongation of the terminal repolarization process by sematilide would enhance the chance of conduction slowing at less complete repolarization levels, which may be associated with a high incidence of TdP induction.

Mechanism of action for N-substituted benzamide-induced apoptosis.

We have analysed the mechanism of action for induction of apoptosis by N-substituted benzamides using declopramide as a lead compound. We show here that declopramide at doses above 250 microM in the mouse 70Z/3 pre-B cell line or in the human promyeolocytic cancer cell line HL60 induced cytochrome c release into the cytosol and caspase-9 activation. The broad spectrum caspase inhibitor zVADfmk and caspase-9 inhibitor zLEDHfmk inhibited apoptosis and improved cell viability when administrated to cells 1 h before exposure to declopramide, whereas the caspase-8 inhibitor zIEDHfmk had less effect. Also, the over expression of Bcl-2 by transfection in 70Z/3 cells inhibited declopramide-induced apoptosis. Prior to the induction of apoptosis, a G(2)/M cell cycle block was induced by declopramide. The cell cycle block was also observed in the presence of broad spectrum caspase inhibitor zVADfmk and in a transfectant expressing high levels of Bcl-2. Furthermore, while p53 was induced in 70Z/3 cells by declopramide, neither the apoptotic mechanism nor the G(2)/M cell cycle block were dependent on p53 activation since both effects were also seen in p53 deficient HL60 cells after addition of declopramide.

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.

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.

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.