Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma.

BACKGROUND: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk. AIM: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. SETTINGS AND DESIGN: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. MATERIALS AND METHODS: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation. RESULTS AND CONCLUSIONS: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.

Immunomodulation of bivalent Newcastle disease DNA vaccine induced immune response by co-delivery of chicken IFN-γ and IL-4 genes.

The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine.

In vivo seizure induction and affinity studies of domoic acid and isodomoic acids-D, -E and -F.

Domoic acid and its isomers are produced via algal blooms and are found in high concentrations in shellfish. Here, we assessed the acute seizurogenic potencies of isomers-D, -E and -F and their binding affinities at heterogeneous populations of KA receptors from rat cerebrum. In addition, binding affinities of all six isomers (Iso-A through -F) were assessed at AMPA receptors. Radioligand displacement studies indicated that the seizurogenic potency of Iso-F (E-configuration) closely correlates with its affinities at both KA and AMPA receptors, whereas isomers-D (Z) and -E (E), which exhibit distinctly lower seizurogenic potencies, are quite weak displacers. Previously observed functional potencies for isomers-A, -B and -C (Sawant et al., 2008) correlated with AMPA receptor affinities observed here. Taken together, these findings call into question previous structure-activity rules. Significantly, in our hands, Iso-D was ten-fold less potent than Iso-F. To further explain observed links between structural conformation and functional potency, molecular modeling was employed. Modeling results closely matched the rank order of potency and binding data observed. We further assessed the efficacy of isomers-D, -E and -F as pharmacological preconditioning agents. Acute preconditioning with low-dose Iso-D, -E or -F, before high-dose DA failed to impart behavioural tolerance. This study has shed new light on structural conformations affecting non-NMDA ionotropic glutamate receptor binding and functional potency, and provides a foundation for future work in areas of AMPA and KA receptor modeling.

Spectral analysis of electrocorticographic activity during pharmacological preconditioning and seizure induction by intrahippocampal domoic acid.

Previously we have shown that low-dose domoic acid (DA) preconditioning produces tolerance to the behavioral effects of high-dose DA. In this study, we used electrocorticography (ECoG) to monitor subtle CNS changes during and after preconditioning. Young adult male Sprague-Dawley rats were implanted with a left cortical electrode, and acute recordings were obtained during preconditioning by contralateral intrahippocampal administration of either low-dose DA (15 pmoles) or saline, followed by a high-dose DA (100 pmoles) challenge. ECoG data were analyzed by fast Fourier transformation to obtain the percentage of baseline power spectral density (PSD) for delta to gamma frequencies (range: 1.25-100 Hz). Consistent with previous results, behavioral analysis confirmed that low-dose DA preconditioning 60 min before a high-dose DA challenge produced significant reductions in cumulative seizure scores and high level seizure behaviors. ECoG analysis revealed significant reductions in power spectral density across all frequency bands, and high-frequency/high-amplitude spiking in DA preconditioned animals, relative to saline controls. Significant correlations between seizure scores and ECoG power confirmed that behavioral analysis is a reliable marker for seizure analysis. The reduction of power in delta to gamma frequency bands in contralateral cortex does not allow a clear distinction between seizure initiation and seizure propagation, but does provide objective confirmation that pharmacological preconditioning by DA reduces network seizure activity.

Evaluation of a capillary blood collection system for screening for hemoglobinopathies in remote areas.

Accurate estimation of hemoglobin (Hbs) A, Hb A(2), Hb F and abnormal Hb is required for diagnosis of hemoglobinopathies and genetic counseling. High pressure liquid chromatography (HPLC) is the most suitable approach available. But for 70% of the rural Indian population, HPLC analysis facilities are not available and screening would require transportation of samples to laboratories in bigger cities. We thus evaluated the feasibility of using a kit designed for measuring Hb A(1c) using capillary blood for collection and preservation of samples over a period of 15 days at different temperatures for screening for hemoglobinopathies. Capillary blood (5 microl) of 90 individuals was collected in the capillary collection system and run on the Variant Hemoglobin Testing System on days 1, 3, 5, 8, 12 and 15 after incubation at 4, 22, 37, 42 and 50 degrees C. The stability of different Hbs varied at different temperatures. The stability was maintained for 12 to 15 days by most of the samples up to 37 degrees C. Hb E was stable for 3 days up to at 37 degrees C and Hb D and Hb Q for 3 days up to 42 degrees C. This capillary blood collection system would have tremendous potential for sample collection and transportation under adverse climatic conditions for screening of hemoglobinopathies in remote areas in different countries.

In vivo seizure induction and pharmacological preconditioning by domoic acid and isodomoic acids A, B and C.

To date, nothing is known of the pharmacological properties of isomers of domoic acid (DA) in vivo in mammals. Here we assessed the acute seizurogenic and toxic properties of DA, isodomoic acids A, B and C (Iso-A, -B, -C), and the therapeutic potential of these compounds as pharmacological preconditioning agents. DA, Iso-A, Iso-B, and Iso-C all produced significant dose-dependent increases in seizure activity following intrahippocampal administration; doses producing half maximal cumulative seizure scores (ED50) were 137 pmol, 171 pmol, 13,000 pmol, and 3150 pmol, respectively. Pharmacological preconditioning with low-dose DA or Iso-A, 60 min before a high test dose of DA produced a significant reduction in seizure scores. In contrast, Iso-B and Iso-C each failed to induce any detectable tolerance to high-dose DA. Radioligand binding indicated a significant correlation between seizurogenic potency and kainate receptor affinity with KIs of 2.4 nM, 4.4 nM, 4990 nM and 170 nM for DA, Iso-A, Iso-B and Iso-C, respectively. Our in vivo results indicate that DA and Iso-A are functionally equipotent in acute seizure induction by direct intrahippocampal administration, while Iso-B and Iso-C are distinctly less potent.

Isodomoic acids A and C exhibit low KA receptor affinity and reduced in vitro potency relative to domoic acid in region CA1 of rat hippocampus.

Several natural isomers of the seizurogenic neurotoxin domoic acid (DA) have been found to occur at up to mg/kg levels in shellfish. The aim of the current study was to assess the neurotoxic potency of isodomoic acids A and C (Iso-A and Iso-C), recently isolated from commercial shellfish. Hippocampal slices were obtained from young adult rats and maintained in a tissue recording chamber. Synaptically evoked population spikes were recorded in region CA1 before and after exposure to DA or its isomers. Both Iso-A and Iso-C produced transient neuronal hyperexcitability followed by a dose-dependent suppression of population spikes, but were, respectively, 4- and 20-fold less potent than DA (spike area: EC50 DA=237 nM; Iso-A=939 nM; Iso-C=4.6 microM). In the hippocampus, DA preconditioning induces tolerance to subsequent DA toxicity. However, in the present study neither Iso-A nor Iso-C were effective as preconditioning agents. Competitive binding studies using homomeric GluR6 kainate (kainic acid, KA) receptors showed the affinity of Iso-A to be 40-fold lower than DA (Ki DA=3.35 nM; Iso-A=130 nM). Together with earlier work showing Iso-C affinity at GluR6 receptors to be 240-fold lower than DA, our results suggest that neuroexcitatory effects of Iso-A in CA1 may involve both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and KA receptors, while Iso-C likely involves the activation of AMPA receptors alone.