Estradiol and dehydroepiandrosterone potentiate levodopa-induced locomotor activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine monkeys

Six monkeys were rendered hemiparkinsonian with a unilateral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These monkeys displayed ipsilateral circling under basal conditions, and after dopaminergic stimulation with levodopa they decreased their ipsilateral circling and started turning to the contralateral side of their lesion. The effect of 17β-estradiol and dehydroepiandrosterone (DHEA) was investigated in these animals. 17β-Estradiol (0.1 mg/kg) added to a threshold dose of levodopa significantly potentiated contralateral circling (mean/30 min) compared to saline or threshold levodopa treatment whereas the duration of circling remained unchanged. DHEA (1–15 mg/kg) alone induced contralateral circling, compared to saline treatment, for 90 min. In addition, DHEA (1–15 mg/kg) potentiated the contralateral circling (mean/30 min) induced by a threshold dose of levodopa and did not change the duration of levodopa circling. A maximal response was observed with 1 or 5 mg/kg of DHEA combined with levodopa depending on the monkey. No correlation was found between the dose for the maximal DHEA response and baseline circling or threshold dose of levodopa. These results suggest that 17β-estradiol or DHEA is able to potentiate locomotor activity of hemiparkinsonian monkeys. The DHEA doses investigated are similar to those presently used in humans. DHEA may be an alternative to 17β-estradiol to modulate dopaminergic activity.     

Inhibition of gastrointestinal motility by MPTP via adrenergic and dopaminergic mechanisms

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was injected intraperitoneally in mice and caused an acute inhibition (of over 60%) of gastrointestinal motility, which was measured by the transit of charcoal. This inhibition was not related to conversion of MPTP to MPP⁺. Administration of the -adrenergic blocker propranolol significantly reduced, but did not completely block, the effect of MPTP. The dopaminergic blocker haloperidol also partly reversed the effects of MPTP. When these blockers were administered together, the action of MPTP was fully blocked. The results indicate that the toxin acted by releasing catecholamines (presumably norepinephrine and dopamine), thereby inhibiting motility.Supported by a grant from the Joint Research Fund of the Hebrew University and Hadassah.     

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.     

RET signaling does not modulate MPTP toxicity but is required for regeneration of dopaminergic axon terminals

Activation of the RET (rearranged during transfection) receptor by glial cell-line-derived neurotrophic factor (GDNF) has been identified as an important differentiation and survival factor for dopaminergic neurons of the midbrain in preclinical experiments. These encouraging results have led to clinical trials of GDNF in patients with Parkinson's disease, which have resulted in conflicting findings. To investigate the potential benefit of Ret-dependent signaling on the challenged dopaminergic system, we tested the effect of tissue-selective ablation of the Ret gene on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice, the most widely used animal model for Parkinson's disease. Ablation of Ret did not modify the MPTP-induced loss of dopaminergic neurons in the substantia nigra pars compacta and the dopaminergic innervation of the striatum at 14 days. However, Ret ablation abolished the regeneration of dopaminergic fibers and terminals, as well as the partial recovery of striatal dopamine concentrations, that was observed in control mice between days 14 and 90 after MPTP treatment. We therefore conclude that RET signaling has no influence on the survival of dopaminergic neurons in the MPTP model of Parkinson's disease but rather facilitates the regeneration of dopaminergic axon terminals.     

Therapeutic effect of a novel anti-parkinsonian agent zonisamide against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)neurotoxicity in mice

We investigated the therapeutic effect of zonisamide against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, using Western blot analysis, immunohistochemistry and behavioral test. Our Western blot analysis and immunohistochemical study showed that the post-treatment with zonisamide prevented significantly dopaminergic cell damage, the depletion of tyrosine-hydroxylase (TH) protein levels and the proliferation of microglia in the striatum and/or substantia nigra 8 days after MPTP treatment. Furthermore, our behavioral study showed that the post-treatment with zonisamide attenuated significantly the motor deficits 7 days after MPTP treatment. These results show that zonisamide has the therapeutic effect in the MPTP model of Parkinson’s disease (PD) in mice. Our study also demonstrates the neuroprotective effect of zonisamide against dopaminergic cell damage after MPTP treatment in mice. Thus our present findings suggest that therapeutic strategies targeted to the activation of TH protein and/or the inhibition of microglial activation with zonisamide may offer a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with PD.     

Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson’s disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP⁺). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.     

Therapeutic Effect of Neuronal Nitric Oxide Synthase Inhibitor (7-Nitroindazole) against MPTP Neurotoxicity in Mice

Effects of neuronal nitric oxide synthase (nNOS) inhibitor (7-nitroindazole), nonselective NOS inhibitor (N^G-nitro-L-arginine methyl ester; L-NAME), and monoamine oxidase inhibitor (pargyline) were studied on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. The mice received four intraperitoneal injections of MPTP at 1-h intervals. A significant depletion in dopamine and DOPAC concentration was observed in the striatum from 1 day after MPTP treatment. The pretreatment of 7-nitroindazole and pargyline, but not L-NAME, dose-dependently protected against MPTP-induced depletion in dopamine content 3 days after MPTP treatment. Our histochemical study also showed that 7-nitroindazole and pargyline can prevent a marked decrease in the nigral cells and a marked increase in astrocytes in striatum 7 days after MPTP treatment. The protective effect of 7-nitroindazole against MPTP-induced dopamine and DOPAC depletion in the striatum was not attenuated by intraperitoneal pretreatment with L-arginine. Furthermore, the posttreatment of 7-nitroindazole or pargyline protected against MPTP-induced depletion of dopamine content. These results demonstrate that the protective mechanism by which 7-nitroindazole counteracts MPTP neurotoxicity in mice may be due not only to inhibition of nNOS, but also to MAO-B inhibition. Furthermore, our study suggests that the posttreatment of 7-nitroindazole and pargyline can prevent a significant decrease in dopamine levels in the striatum of MPTP-treated mice. These findings have important implications for the therapeutic time window and choice of nNOS or MAO inhibitors in patients with Parkinson's disease.     

Effects of estrogens on striatal damage after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in male and female mice

Nurr1, NGFI-B, and Nor1 form the NR4A subfamily of orphan nuclear receptors. The NR4A receptors are immediate early genes that can be rapidly induced in response to a variety of stimuli in many cell types, for example, in osteoblasts. Nurr1 regulates the differentiation of osteoblasts and the expression of several osteoblastic genes. Fibroblast growth factor 8b (FGF-8b) regulates osteoblastic differentiation. We show here that treatment of preosteoblastic MC3T3-E1 cells or mouse bone marrow mesenchymal cells with FGF-8b induces the expression of NR4A receptors rapidly and in a dose-dependent manner. This induction involves mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI-3K), and protein kinase C (PKC) pathways. FGF-8b stimulates the proliferation of MC3T3-E1 cells. This effect is enhanced by overexpression of Nurr1 and NGFI-B whereas it is abolished by a dominant negative Nurr1 variant. In conclusion, FGF-8b induces the expression of NR4A orphan nuclear receptors that are involved in mediating the growth promoting effect of FGF-8b in osteoblasts.     

Alterations of gene expression of sodium channels in dorsal root ganglion neurons of estrogen receptor knockout (ERKO) mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Estrogen receptors (ERα and ERβ) mediate the neuroprotection of estrogens against MPTP-induced striatal dopamine (DA) depletion. Pain is an important and distressing symptom in Parkinson's disease (PD). Voltage-gated sodium channels in sensory neurons are involved in the development of neuropathic pain. In this study, MPTP caused changes in nociception and alterations of gene expression of voltage-gated sodium channels in dorsal root ganglion (DRG) neurons in ER knockout (ERKO) mice were investigated. We found that administration of MPTP (11 mg/kg) to WT mice led to an extensive depletion of DA and its two metabolites, αERKO mice were observed to be more susceptible to MPTP toxicity than βERKO or WT mice. In addition, we found that the mRNA levels of TTX-S and TTX-R sodium channel subtypes were differentially affected in MPTP-treated WT animals. The MPTP-induced up-regulation of Nav1.1 and Nav1.9, down-regulation of Nav1.6 in DRG neurons may be through ERβ, up-regulation of Nav1.7 and down-regulation of Nav1.8 are dependent on both ERα and ERβ. Therefore, the MPTP-induced alterations of gene expression of sodium channels in DRG neurons could be an important mechanism to affect excitability and nociceptive thresholds, and the ERs appear to play a role in nociception in PD.     

Therapeutic effect of a novel anti-parkinsonian agent zonisamide against MPTP (1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine) neurotoxicity in mice

We investigated the therapeutic effect of zonisamide against 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, using Western blot analysis, immunohistochemistry and behavioral test. Our Western blot analysis and immunohistochemical study showed that the post-treatment with zonisamide prevented significantly dopaminergic cell damage, the depletion of tyrosine-hydroxylase (TH) protein levels and the proliferation of microglia in the striatum and/or substantia nigra 8 days after MPTP treatment. Furthermore, our behavioral study showed that the post-treatment with zonisamide attenuated significantly the motor deficits 7 days after MPTP treatment. These results show that zonisamide has the therapeutic effect in the MPTP model of Parkinson’s disease (PD) in mice. Our study also demonstrates the neuroprotective effect of zonisamide against dopaminergic cell damage after MPTP treatment in mice. Thus our present findings suggest that therapeutic strategies targeted to the activation of TH protein and/or the inhibition of microglial activation with zonisamide may offer a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with PD.     

肾脏D_5多巴胺受体高表达参与了血管紧张素Ⅱ1型受体基因敲除小鼠低血压的发生

目的肾脏血管紧张素Ⅱ1型受体(AT1R)的保钠潴水功能部分可以解释AT1R基因敲除(AT1A-/-)小鼠血压降低的原因,然而,对于是否有AT1R以外的机制参与则研究较少。既往的研究间接提示D5多巴胺受体和AT1R之间存在相互抑制作用,因而在本实验中,将验证D5受体的因素是否参与AT1A-/-小鼠低血压的发生。方法在比较AT1A-/-小鼠和其对照(AT1A / )小鼠血压、尿钠排泄、肾脏D5受体表达的基础上,利用Wistar-Kyoto(WKY)大鼠的肾近曲小管(RPT)细胞株和D5受体转染HEK293细胞为研究对象,研究AT1R和D5受体之间的交互影响,探讨AT1A-/-小鼠血压降低的原因。结果与AT1A / 小鼠相比,低盐饮食的状况下AT1A-/-小鼠血压较低、尿钠排泄能力增强,肾脏D5受体表达量增加。在WKY大鼠肾近曲小管的细胞上,刺激AT1R可特异地抑制D5受体的表达;反过来,刺激D5受体转染HEK293细胞的D5受体也可抑制AT1R的表达。结论D5受体的高表达可能参与了AT1A-/-小鼠血压降低的发生机制。     

Melatonin enhances L‐DOPA therapeutic effects,helps to reduce its dose,and protects dopaminergic neurons in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced parkinsonism in mice

L‐3,4‐dihydroxyphenylalanine (L‐DOPA) reduces symptoms of Parkinson's disease (PD), but suffers from serious side effects on long‐term use. Melatonin (10–30 mg/kg, 6 doses at 10 hr intervals) was investigated to potentiate L‐DOPA therapeutic effects in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced parkinsonism in mice. Striatal tyrosine hydroxylase (TH) immunoreactivity, TH, and phosphorylated ser 40 TH (p‐TH) protein levels were assayed on 7th day. Nigral TH‐positive neurons stereology was conducted on serial sections 2.8 mm from bregma rostrally to 3.74 mm caudally. MPTP caused 39% and 58% decrease, respectively, in striatal fibers and TH protein levels, but 2.5‐fold increase in p‐TH levels. About 35% TH neurons were lost between 360 and 600 μm from 940 μm of the entire nigra analyzed, but no neurons were lost between 250 μm rostrally and 220 μm caudally. When L‐DOPA in small doses (5–8 mg/kg) failed to affect MPTP‐induced akinesia or catalepsy, co‐administration of melatonin with L‐DOPA attenuated these behaviors. Melatonin administration significantly attenuated MPTP‐induced loss in striatal TH fibers (82%), TH (62%) and p‐TH protein (100%) levels, and nigral neurons (87–100%). Melatonin failed to attenuate MPTP‐induced striatal dopamine depletion. L‐DOPA administration (5 mg/kg, once 40 min prior to sacrifice, p.o.) in MPTP‐ and melatonin‐treated mice caused significant increase in striatal dopamine (31%), as compared to L‐DOPA and MPTP‐treated mice. This was equivalent to 8 mg/kg L‐DOPA administration in parkinsonian mouse. Therefore, prolonged, effective use of L‐DOPA in PD with lesser side effects could be achieved by treating with 60% lower doses of L‐DOPA along with melatonin.     

The Effect of Intensive Insulin Therapy on the Insulin-regulatable Glucose Transporter (GLUT4) Expression in Skeletal Muscle in Type 1 Diabetes

Studies in normal man and rodents have demonstrated that the expression of the dominant glucose transporter in skeletal muscle, GLUT4, is regulated by insulin at supraphysiological circulating levels. The present study was designed to determine whether intensified insulin replacement therapy for 24 h given to patients with Type 1 diabetes in poor metabolic control was associated with an adaptive regulation of GLUT4 mRNA and protein levels in vastus lateralis muscle. Nine Type 1 diabetic patients with a mean HbA_1c of 10.3% were included in the protocol. After intensified treatment with soluble insulin for 24 h the fasting plasma glucose concentration decreased from 20.8 ± 2.3 (SD) to 8.7 ± 2.3 mmol 1^?1 whereas the fasting serum insulin level increased from 0.06 ± 0.02 to 0.17 ± 0.09 nmol 1^?1 However, despite a 2.8-fold increase in serum insulin levels and more than a halving of the plasma glucose concentration for at least 15 h no significant alterations occurred in the amount of GLUT4 protein (0.138 ± 0.056, poor control vs 0.113 ± 0.026 arb. units, improved control, p = 0.16) or GLUT4 mRNA (96432 ± 44985, poor control vs 81395 ± 25461 arb. units, improved control, p = 0.54). These results suggest, that in spite of evidence that high insulin levels affect GLUT4 expression in muscle, changes in serum insulin within the physiological range do not play a major role in the short-term regulation of GLUT4 expression in Type 1 diabetic patients.     

Crucial Role of c-Jun NH2-Terminal Kinase 1 (JNK1) in Cold-Restraint Stress-Induced Gastric Lesions in Mice

c-Jun NH2-terminal kinase 1 /JNK1, is activated in response to a broad array of cellular stresses. We investigated the role of JNK1 in the pathophysiology of cold-restraint stress-induced gastric lesions in mice. B6/129, wild type (WT) mice, or mutant mice deficient in Jnk1 (Jnk1-/- mice) were exposed to cold-restraint stress for different time periods. Gastric lesions were identified and quantitated by morphometric analysis. JNK1 activity in mucosal homogenates was quantitated by immunoprecipitation and in-vitro kinase assays. JNK1 expression and Akt activation were assessed by Western blots with anti-JNK1 and anti-phospho Akt antibodies, respectively. Gastric mucosal homogenates from Jnk1-/- mice exhibited no significant expression of JNK1 and no detectable level of JNK1 activation. Exposure of WT mice to cold-restraint stress led to the development of significant gastric lesions and to a greater than three-fold induction in JNK1 activity, while no lesions were detected in the gastric mucosa of Jnk1-/- mice. Since cold-restraint stress-induced gastric lesions involve the activation of cholinergic pathways, we tested the effect of atropine on both the development of gastric lesions and JNK1 activation. Pretreatment of WT mice with atropine completely inhibited both cold-restraint stress-induced lesions and JNK1 activation. Cold-restraint stress induced protein kinase B/Akt to a similar level in the gastric mucosa of both WT and Jnk1-/- mice indicating the integrity of other signaling pathways. JNK1 plays a key role in the development of cold-restraint stress-induced gastric lesions in mice through the activation of cholinergic, atropine sensitive pathways.     

Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4-/- mouse model

AIM: To study the interleukin-1(IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4~(-/-)(Abcb4~(-/-)) mouse model.METHODS: Female and male Abcb4~(-/-) mice from 6 to 13 mo of age were analysed for the degree of cholestasis(liver serum tests), extent of liver fibrosis(hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction(q PCR)]. For in vivo experiments, murine hepatic stellate cells(HSCs) were isolated via pronasecollagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/m L IL-1β with or without 2.5 μg/m L Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the Brd U assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis(FDH) assay. In vivo 8-wk-old Abcb4~(-/-) mice with an already fully established hepatic phenotype were treated with Anakinra(1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, q PCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4~(-/-) animals as defined by a lower hydroxyproline content(274 ± 64 μg/g vs 436 ± 80 μg/g liver, respectively; n = 13-15; P 0.001; MannWhitney U-test) and lower m RNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1(TIMP)(1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 m RNA expression(1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P 0.05; Mann-Whitney U-test) and significantly lower IL-1β m RNA expression levels(1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P 0.001; Mann-Whitney U-test). No gender differences in the serum liver parameters [bilirubin; alanine aminotransferase(ALT); aspartate aminotransferase and alkaline phosphatase(AP)] were found. In vitro, the administration of IL-1β resulted in a significant increase in HSC proliferation [0.94 ± 0.72 arbitrary units(A.U.) in untreated controls, 1.12 ± 0.80 A.U. at an IL-1β concentration of 0.1 ng/m L and 1.18 ± 0.73 A.U. at an IL-1β concentration of 1 ng/m L in samples from n = 6 donor animals; P 0.001; analyses of variance(ANOVA)]. Proliferation was reduced significantly by the addition of 2.5 μg/m L Anakinra(0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1β concentration of 0.1 ng/m L, and 0.91 ± 0.69 A.U. at an IL-1β concentration of 1 ng/m L; in samples from n = 6 donor animals; P 0.001; ANOVA) suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist. The FDH assay showed this dose to be non-toxic in HSCs. In vivo, Anakinra had no effect on the hepatic hydroxyprolinecontent, liver serum tests(ALT and AP) and profibrotic(collagen 1α1, collagen 1α2, transforming growth factor-β, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2(MMP2), MMP9 and MMP13 ] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1β and F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION: IL-1β expression is associated with the degree of liver fibrosis in Abcb4~(-/-) mice and promotes HSC proliferation. IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4~(-/-) mice.     

Features of the Preparation and Luminescence of Langmuir-Blodgett Films Based on the Tb(III) Complex with 3-Methyl-1-phenyl-4-stearoylpyrazol-5-one and 2,2′-Bipyridine

In this study, we investigated the effect of terbium ions (Tb³⁺⁾ on the subphases of the limiting area of the molecule for the complex compound (CC) TbL₃∙bipy (where HL is 3-methyl-1-phenyl-4-stearoylpyrazol-5-one and bipy is 2,2′-bipyridine). We examined the Langmuir monolayer and the change in the luminescence properties of TbL₃∙bipy-based Langmuir-Blodgett films (LBFs). The analysis of the compression isotherms, infrared, and luminescence spectra of TbL₃∙bipy LBFs was performed by varying the concentration of Tb³⁺ in the subphases. Our results demonstrate the partial dissociation of the CC at concentrations of C(Tb³⁺) < 5 × 10⁻⁴ M.     

Genetic polymorphisms in platelet-related proteins and coronary artery disease: investigation of candidate genes, including N-acetylgalactosaminyltransferase 4 (GALNT4) and sulphotransferase 1A1/2 (SULT1A1/2)

Background Both platelet function and heart disease show strong genetic components, many of which remain to be elucidated. Materials and methods The roles of candidate polymorphisms in ten platelet-associated genes were compared between 1,237 Acute Coronary Syndrome (ACS) cases (with myocardial infarction and unstable angina) and 386 controls, from an Irish Caucasian population. Additionally, 361 stable angina patients were investigated. Two genes of interest were followed up in a separate Irish study of 1,484 individuals (577 with IHD and 907 unaffected). Results The GALNT4 (N-acetyl galactosaminyl transferase 4) 506I allele was significantly underrepresented in ACS (OR = 0.66, CI = 0.52-0.84; P = 0.001; P = 0.01 after correction for multiple testing), while the SULT1A1 (Sulphotransferase 1A1) 213H allele was associated with risk of ACS (OR = 1.37, CI = 1.08-1.74; P = 0.01; P = 0.1 after correction for multiple testing). Subsequent genotyping of further SNPs in GALNT4 in the family-based (IHD) group revealed that the 506I allele showed the same trend towards protecting against ACS but the haplotypic test over the four commonest haplotypes was not significant (P = 0.55). In contrast, the SULT1A1/SULT1A2 gene complex showed suggestive haplotypic association in the family-based study (P = 0.07), with the greatest increase in risk conferred by the SULT1A2 235T allele (P = 0.025). Conclusion We have identified two risk genes for cardiovascular disease, one of whose (GALNT4) effects may be on either platelet or endothelial function through modifications of PSGL1 or other important glycosylated proteins. The role of sulphotransferases (SULT1A1/2) in cardiovascular disease requires further exploration. Further validation of cardiovascular risks conferred by both genes in other populations (including gene copy number variation) is warranted.     

Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections

Open reading frame 2 proteins (ORF2) from swine (genotype 4, S-ORF2) and human (genotype 1, H-ORF2) hepatitis E virus (HEV) having 91.4% identity at amino acid level were expressed using baculovirus expression system. Comparison of ELISAs based on the two proteins yielded identical results when sequential serum samples from monkeys and pigs experimentally infected with genotypes 1 and 4 HEV, respectively, were tested. Samples from patients (n = 258) suffering from non-A, non-B hepatitis during outbreaks of the disease and 180 sera from apparently healthy children were screened by H-ORF2-, S-ORF2-based ELISAs and Genelabs ELISA, a widely used commercial test for HEV diagnosis. Specificity of all three tests in detecting IgM and IgG antibodies in healthy children was comparable. Excellent correlation was noted in detecting both IgM (98.7% concordance) and IgG (97.7% concordance) anti-HEV antibodies when H-ORF2 and S-ORF2 ELISAs were compared. When compared with Genelabs ELISA, both H-ORF2 and S-ORF2 ELISAs identified 34 and 18 additional positives, respectively, in IgM and IgG anti-HEV tests showing comparatively less sensitivity of the commercial assay. The concordance of Genelabs ELISA in IgM detection was 86.4% and 85.6%, respectively, with H-ORF2 and S-ORF2 ELISAs. The concordance between Genelabs ELISA and H-ORF2 decreased further to 73.6% when 129 human samples from recent HEV epidemics (2002-2004) were tested for IgM. Similar results were obtained when sequential samples from 11 hepatitis E patients were examined. Screening of serum samples from 137 sporadic non-A, non-B hepatitis cases further confirmed the superiority of the H-ORF2 and S-ORF2 ELISAs. All 36/137 HEV-RNA-positive samples from sporadic cases belonged to genotype 1 confirming absence/rarity of type 4 human infections. H-ORF2 and S-ORF2 antigens were swappable in ELISAs for detecting both genotypes 1 and 4 HEV infections.