Lack of association of rs3798220 with small apolipoprotein(a) isoforms and high lipoprotein(a) levels in East and Southeast Asians.

OBJECTIVE: The variant allele of rs3798220 in the apolipoprotein(a) gene (LPA) is used to assess the risk for coronary artery disease (CAD) in Europeans, where it is associated with short alleles of the Kringle IV-2 (KIV-2) copy number variation (CNV) and high lipoprotein(a) (Lp(a)) concentrations. No association of rs3798220 with CAD was detected in a GWAS of East Asians. Our study investigated the association of rs3798220 with Lp(a) concentrations and KIV-2 CNV size in non-European populations to explain the missing association of the variant with CAD in Asians. METHODS: We screened three populations from Africa and seven from Asia by TaqMan Assay for rs3798220 and determined KIV-2 CNV sizes of LPA alleles by pulsed-field gel electrophoresis (PFGE). Additionally, CAD cases from India were analysed. To investigate the phylogenetic origin of rs3798220, 40 LPA alleles from Chinese individuals were separated by PFGE and haplotyped for further SNPs. RESULTS: The variant was not found in Africans. Allele frequencies in East and Southeast Asians ranged from 2.9% to 11.6%, and were very low (0.15%) in CAD cases and controls from India. The variant was neither associated with short KIV-2 CNV alleles nor elevated Lp(a) concentrations in Asians. CONCLUSION: Our study shows that rs3798220 is no marker for short KIV-2 CNV alleles and high Lp(a) in East and Southeast Asians, although the haplotype background is shared with Europeans. It appears unlikely that this SNP confers atherogenic potential on its own. Furthermore, this SNP does not explain Lp(a) attributed risk for CAD in Asian Indians.

Murine model: maternal administration of stem cells for prevention of prematurity.

OBJECTIVE: Using a mouse model of intrauterine inflammation, we have demonstrated that exposure to inflammation induces preterm birth and perinatal brain injury. Mesenchymal stem cells (MSCs) have been shown to exhibit immunomodulatory effects in many inflammatory conditions. We hypothesized that treatment with human adipose tissue-derived MSCs may decrease the rate of preterm birth and perinatal brain injury through changes in antiinflammatory and regulatory milieu. STUDY DESIGN: A mouse model of intrauterine inflammation was used with the following groups: (1) control; (2) intrauterine inflammation (lipopolysaccharide); and (3) intrauterine lipopolysaccharide+intraperitoneal (MSCs). Preterm birth was investigated. Luminex multiplex enzyme-linked immunosorbent assays were performed for protein levels of cytokines in maternal and fetal compartments. Immunofluorescent staining was used to identify and localize MSCs and to examine microglial morphologic condition and neurotoxicity in perinatal brain. Behavioral testing was performed at postnatal day 5. RESULTS: Pretreatment with MSCs significantly decreased the rate of preterm birth by 21% compared with the lipopolysaccharide group (P<.01). Pretreatment was associated with increased interleukin-10 in maternal serum, increased interleukin-4 in placenta, decreased interleukin-6 in fetal brain (P<.05), decreased microglial activation (P<.05), and decreased fetal neurotoxicity (P<.05). These findings were associated with improved neurobehavioral testing at postnatal day 5 (P<.05). Injected MSCs were localized to placenta. CONCLUSION: Maternally administered MSCs appear to modulate maternal and fetal immune response to intrauterine inflammation in the model and decrease preterm birth, perinatal brain injury, and motor deficits in offspring mice.

Mouse model of intrauterine inflammation: sex-specific differences in long-term neurologic and immune sequelae.

Preterm infants, especially those that are exposed to prenatal intrauterine infection or inflammation, are at a major risk for adverse neurological outcomes, including cognitive, motor and behavioral disabilities. We have previously shown in a mouse model that there is an acute fetal brain insult associated with intrauterine inflammation. The objectives of this study were: (1) to elucidate long-term (into adolescence and adulthood) neurological outcomes by assessing neurobehavioral development, MRI, immunohistochemistry and flow cytometry of cells of immune origin and (2) to determine whether there are any sex-specific differences in brain development associated with intrauterine inflammation. Our results have shown that prenatal exposure appeared to lead to changes in MRI and behavior patterns throughout the neonatal period and during adulthood. Furthermore, we observed chronic brain inflammation in the offspring, with persistence of microglial activation and increased numbers of macrophages in the brain, ultimately resulting in neuronal loss. Moreover, our study highlights the sex-specific differences in long-term sequelae. This study, while extending the growing literature of adverse neurologic outcomes following exposure to inflammation during early development, presents novel findings in the context of intrauterine inflammation.

Targeting chelatable iron as a therapeutic modality in Parkinson's disease.

AIMS: The pathophysiological role of iron in Parkinson's disease (PD) was assessed by a chelation strategy aimed at reducing oxidative damage associated with regional iron deposition without affecting circulating metals. Translational cell and animal models provided concept proofs and a delayed-start (DS) treatment paradigm, the basis for preliminary clinical assessments. RESULTS: For translational studies, we assessed the effect of oxidative insults in mice systemically prechelated with deferiprone (DFP) by following motor functions, striatal dopamine (HPLC and MRI-PET), and brain iron deposition (relaxation-R2*-MRI) aided by spectroscopic measurements of neuronal labile iron (with fluorescence-sensitive iron sensors) and oxidative damage by markers of protein, lipid, and DNA modification. DFP significantly reduced labile iron and biological damage in oxidation-stressed cells and animals, improving motor functions while raising striatal dopamine. For a pilot, double-blind, placebo-controlled randomized clinical trial, early-stage Parkinson's patients on stabilized dopamine regimens enrolled in a 12-month single-center study with DFP (30 mg/kg/day). Based on a 6-month DS paradigm, early-start patients (n=19) compared to DS patients (n=18) (37/40 completed) responded significantly earlier and sustainably to treatment in both substantia nigra iron deposits (R2* MRI) and Unified Parkinson's Disease Rating Scale motor indicators of disease progression (p<0.03 and p<0.04, respectively). Apart from three rapidly resolved neutropenia cases, safety was maintained throughout the trial. INNOVATION: A moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality for PD. CONCLUSIONS: The therapeutic features of a chelation modality established in translational models and in pilot clinical trials warrant comprehensive evaluation of symptomatic and/or disease-modifying potential of chelation in PD.

Huntingtin inclusion bodies are iron-dependent centers of oxidative events.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant.

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.

Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels.

Small stress proteins [small heat shock proteins (sHsps)] are molecular chaperones that modulate the ability of cells to respond to oxidative stress. The current knowledge concerning the protective mechanism generated by the expression of mammalian heat shock protein-27 (Hsp27) that allows cells to increase their resistance to oxidative stress is presented. We describe the effects mediated by Hsp27 expression toward crucial enzymes such as glucose-6-phosphate dehydrogenase and glutathione reductase that uphold glutathione in its reduced form. New data are presented showing that the expression of sHsps correlates with a drastic decrease in the intracellular level of iron, a catalyzer of hydroxyl radical (OH( . )) generation. A decreased ability of sHsps expressing cells to concentrate iron will therefore end up in a decreased level of oxidized proteins. In addition, we propose a role of Hsp27 in the presentation of oxidized proteins to the proteasome degradation machinery. We also present an analysis of several Hsp27 mutants that suggests that the C-terminal part of this stress protein is essential for its protective activity against oxidative stress.

Cytotoxic effects induced by oxidative stress in cultured mammalian cells and protection provided by Hsp27 expression.

There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.