Cardiac 5-HT(4) serotoninergic receptors, 52kD SSA/Ro and autoimmune-associated congenital heart block

It was recently reported that sera from patients with systemic lupus erythematosus contain antibodies reactive with the second extracellular loop of the serotoninergic 5-HT(4) receptor expressed in the human heart. This antibody response was associated with antibodies to 52kD SSA/Ro, a reactivity prevalent in mothers of children with congenital heart block (CHB). The current study was undertaken to determine whether the 5-HT(4) receptor is a target of the immune response in these mothers. Initial experiments demonstrated mRNA expression of the 5-HT(4) receptor in the human foetal atrium. Electrophysiologic studies established that human foetal atrial cells express functional 5-HT(4) receptors. Sera from 116 mothers enrolled in the Research Registry for Neonatal Lupus, whose children have CHB, were evaluated. Ninety-nine (85%) of these maternal sera contained antibodies to SSA/Ro, 84% of which were reactive with the 52kD SSA/Ro component by immunoblot. None of the 116 sera were reactive with the peptide spanning aa165-185 of the serotoninergic receptor. Rabbit antisera which recognized this peptide did not react with 52kD SSA/Ro or peptide aa365-382 in the C terminus. Although 5-HT(4) receptors are present and functional in the human foetal heart, maternal antibodies to the 5-HT(4) receptor are not associated with the development of CHB.     

Autoantibodies against the serotoninergic 5-HT4 receptor and congenital heart block: a reassessment

The specificity of autoantibodies against the serotoninergic 5-HT4 receptor in congenital heart block has led to conflicting observations. In order to clarify the situation, a collaborative effort was undertaken to discover the reasons for these discrepancies and to reassess the importance of such autoantibodies by making use of the Research Registry for Neonatal Lupus. Sera from 128 patients (101 anti-SSA/Ro52 positive mothers among which 74 have children with congenital heart block (CHB), 9 anti-SSA/Ro52 negative patients of which 1 had a child with heart block and 18 healthy donors) were assessed in a single blind test using an ELISA coated with a 5-HT4 receptor-derived peptide. Discrepancies between previous observations in our two groups could be ascribed to small differences in the set up of the assay. Of the 75 sera from mothers of children with CHB, 12 were reactive with the 5-HT4 peptide. Four sera among which three were from 35 Ro52 negative mothers without affected children as well as 2 in the 18 controls were positive. Interestingly, in 1 mother with an isolated child with CHB but who had no detectable anti-SSA/Ro52 antibodies and 1 mother with a child with a structural heart block and no detectable antibodies to any component of SSA/Ro, reactivity with the 5-HT4 receptor was noted. While 5-HT4 receptor autoantibodies do not have the predictive value of anti-Ro52 autoantibodies, the presence of these antibodies in a minor subset of mothers whose children have CHB suggests an additional risk factor which may contribute to the pathogenesis of disease.     

Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block

Autoantibodies against amino acid 200-239 (p200) in the predicted leucine zipper region of the Ro52 protein are associated with congenital heart block, a potentially fatal condition that may affect fetuses of women with Ro52 autoantibodies. To allow detailed studies of the antibodies associated with congenital heart block, B-cell derived combinatorial antibody libraries from patients were screened for Ro52 and p200 specific antibody clones. Two human monoclonal anti-p200 antibody fragments, S3A8 and M4H1, were isolated and analysed with regard to VHand VL gene utilization, somatic mutations and binding properties. Both identified clones recognized recombinant and native intact Ro52, and reacted only with p200 in a set of related Ro52 peptides. The specificity and affinity was confirmed by biosensor measurements. Structural analysis of overlapping peptides revealed increased helicity in the p200 peptide compared to non-recognized peptides, indicating epitope conformation as essential for antibody binding. Both monoclonals produced punctate nuclear and diffuse cytoplasmic staining in human and mouse cell lines. The identified antibodies, which react specifically with the leucine zipper structure of Ro52, will be valuable in further exploration of the mechanisms operating during development of Ro52 antibody-associated congenital heart block.     

Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor

We have previously suggested that the recognition of a cross-reactive epitope on the 5-HT4 receptor and the 52-kDa SSA/Ro protein by serotonin-antagonizing autoantibodies could explain the electrophysiological symptoms of congenital heart block in neonatal lupus. To confirm this hypothesis, we immunized female mice with four synthetic peptides corresponding to the recognized epitopes. All mice developed anti-peptide antibodies, which cross-reacted with the Ro52 and 5-HT4 receptor peptides and recognized both cognate proteins. Peptide-immune mice were mated. The pups from mice immunized with the Ro52 peptides had no symptoms of neonatal lupus apart from bradycardia. However, pups from mice immunized with the 5-HT4 receptor peptides and bradycardia, atrioventricular block of type I or II, longer QT intervals, skin rashes and neuromotor problems. The 5-HT4 receptor was detectable in the different fetal tissues affected (heart, skin and brain) by immunohistochemistry. Hearts from diseased pups were less developed and showed disorganized myocardial hyperplasia, compared to the normal littermates. These results demonstrate that the serotoninergic 5-HT4 receptor is the antigenic target of physiopathological autoantibodies in neonatal lupus.     

Negative symptoms of schizophrenia could explain discrepant data on the association between the 5-HT2A receptor gene and response to antipsychotics

Pharmacogenetic studies assessing the role of 5-HT(2A) receptor gene in antipsychotic efficacy yielded conflicting data. Phenotypical heterogeneity of schizophrenia might explain such discrepancies. For example, negative symptoms are known to reflect severity of illness and to restrain therapeutic response. On this basis, we re-assessed the possible influence of the -1438A/G polymorphism of the 5-HT(2A) receptor gene on the clinical efficacy of atypical antipsychotics with focus on several relevant dimensions. One hundred and sixteen French schizophrenic subjects treated for at least 1 month by atypical antipsychotics were screened for treatment response according to the May and Dencker scale. Gender, age at onset, duration and severity of illness, intensity of negative and positive symptoms at discharge were investigated. The intensity of negative symptoms at discharge was the only variable explaining May and Dencker score (p < 0.001), and was significantly associated with the AA genotype of the -1438A/G polymorphism of the 5-HT(2A) receptor gene (p = 0.03). However, the A allele was not independently associated with refractoriness to atypical antipsychotics. Accordingly, the score reached in the Scale for the Assessment of Negative Symptoms (SANS) appeared as a confounding factor between therapeutic response and the -1438A/G polymorphism of the 5-HT(2A) receptor gene, at least in our sample. This data indicate that negative symptoms are worth being systematically assessed in pharmacogenetic studies aimed at analysing candidate genes in schizophrenia.     

Successful preventive treatment of congenital heart block during pregnancy in a woman with systemic lupus erythematosus and anti-Sj?gren's syndrome A/Ro antibody.

Congenital heart block (CHB) that is a manifestation of neonatal lupus syndrome (NLS) carries a poor prognosis. The treatment response of established heart block in NLS is usually unsatisfactory. Preventive treatment during pregnancy, however, before the critical period of cardiac development, can prevent the development of CHB. A Taiwanese woman with systemic lupus erythematosus (SLE) was positive for anti-Sj?gren's syndrome A (SSA)/Ro antibody. Her first pregnancy resulted in intra-uterine fetal death. Her second pregnancy resulted in CHB, despite dexamethasone treatment, and neonatal death at age 1 day despite pacemaker implantation. During her third pregnancy, dexamethasone was given starting at week 10, azathioprine at week 18, and plasmapheresis was performed every other day for 5 times starting at week 20 of gestation. Cesarean section was performed due to oligohydramnion at week 31 of gestation and a healthy girl was delivered. This case suggests that judicious use of fluorinated glucocorticoids, immunosuppressants, and plasmapheresis may prevent development of CHB in pregnant women with SLE who are anti-SSA/Ro antibody positive and have previous children with CHB.     

Antibodies to amino acid 200-239 (p200) of Ro52 as serological markers for the risk of developing congenital heart block

Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to correlate with development of congenital heart block. The aim of the present study was to evaluate the clinical relevance and predictive value of p200-antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and American women were included in the study. Sera originated from 202 mothers with an infant affected by second- or third-degree atrioventricular block (AVB), 177 mothers with rheumatic disease having infants with normal heart rate and female blood donors (n = 136). A novel serological assay for Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3.8% respectively was developed. Mothers of children affected by AVB II-III had significantly higher p200-antibody levels than mothers with rheumatic disease having children with normal heart rate (P < 0.001). In the Swedish cohort, a distinction between foetuses with normal conduction, AVB I, AVB II and III was possible. A significant difference in anti-p200 levels between AVB I and AVB II-III groups compared with foetuses with normal conduction (P < 0.05 and P < 0.01) was observed. Using p200-antibodies as a second step analysis in Ro52-positive pregnancies increased the positive predictive value for foetal cardiac involvement (AVB I, II or III) from 0.39 (0.27-0.51) to 0.53 (0.37-0.68). In conclusion, Ro52 p200-antibodies may occur in women with unaffected children, but levels are significantly higher in mothers of children with congenital heart block and are suggested as a relevant marker in evaluating the risk for foetal AV block.     

Effects of the 5-HT4 receptor agonist RS67333 and paroxetine on hippocampal extracellular 5-HT levels

The 5-HT₄ receptor modulates activity of serotonergic neurons and is a new potential target for antidepressant treatment. This microdialysis study evaluated the effect of the 5-HT₄ receptor agonist, RS67333, on extracellular serotonin (5-hydroxytryptamine, 5-HT) and 5-HIAA levels in rat ventral hippocampus during chloral hydrate anaesthesia, and explored the ability of RS67333 to augment the effect of the selective serotonin reuptake inhibitor paroxetine. The effect of RS67333 was examined after acute and subchronic (3 days) administration. Acute RS67333 (1.5 mg/kg i.v.) had no effect on extracellular 5-HT or 5-HIAA levels, while acute paroxetine (0.5 mg/kg i.v.) increased 5-HT levels by 299 ± 16% and decreased 5-HIAA levels by 25 ± 4%. Administration of RS67333 80 min after paroxetine caused an additional transient increase in 5-HT levels (to 398 ± 52% of baseline). Subchronic RS67333 administration (1.5 mg/kg i.p.) increased basal 5-HT levels by 73 ± 15% and decreased 5-HIAA levels by 27 ± 13%. In conclusion, the 5-HT₄ receptor agonist RS67333 augmented the acute effect of paroxetine on extracellular 5-HT levels in the ventral hippocampus, and after 3 days increased basal hippocampal 5-HT levels.     

Local infusion of the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione does not block D1 dopamine receptor-mediated increases in immediate early gene expression in the dopamine-depleted striatum

Administration of selective agonists of D1 dopamine receptors increases immediate early gene expression in striatal neurons, a response which is particularly robust in the dopamine-depleted striatum. Although interactions between dopamine and glutamate receptor-mediated responses in striatal neurons have been demonstrated in a number of experimental paradigms, our previous findings indicate that N-methyl-D-aspartate antagonists do not block D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum. In the present study, we therefore examined interactions between D1 dopamine receptors and the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate subtypes of glutamate receptor by determining whether striatal infusion of the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione would block D1 receptor-mediated induction of the immediate early genes c-fos and zif268 in the dopamine-depleted striatum. Striatal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (1 mM) completely blocked (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate-induced c-fos and zif268 expression. However, 6-cyano-7-nitroquinoxaline-2,3-dione (1 microM-1 mM) did not significantly affect induction of c-fos and zif268 by D1 receptor stimulation (SKF 38393, 2 mg/kg, i.p.) in the dopamine-depleted striatum. To more generally block excitatory input, tetrodotoxin (10 microM) was infused into the striatum of rats receiving a D1 agonist. Local infusion of tetrodotoxin had minimal effect on induction of c-fos and zif268 in the dopamine-depleted striatum. In contrast, tetrodotoxin abolished induction of c-fos and zif268 messenger RNAs by the D2 antagonist eticlopride (0.5 mg/kg, i.p.) in both intact rats and dopamine-depleted rats receiving continuous D2 agonist treatment (quinpirole, 0.5 mg/kg/day). The results indicate that D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum occurs by mechanisms that are independent of excitatory input through (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors.     

Effects of N-methyl-d-aspartate (dizocilpine) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat

The present study was undertaken to examine the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3-10 mg/kg, i.p.), a competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34-53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1-3 mg/kg, i.p.), a non-competitive N-methyl-D-aspartate receptor antagonist, also significantly decreased the urine release (62-82%) and increased residual volume (180-230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%. These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.