Human platelet antigen genotyping of platelet donors in southern Brazil.

Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

Association between HLA-DR4 haplotypes and tuberculin skin test response in the Aché population.

The human leukocyte antigen (HLA) system has a major role in the regulation of the immune response as it is involved in the defense against pathogens. Evidence for association with tuberculosis (TB) is more consistent for class II than for class I HLA genes. TB is important among indigenous peoples in South America, not only because of its historical role in regional depopulation, but also because it is still widespread. The aim of this study was to evaluate the association of HLA class II alleles, haplotypes and genotypes and tuberculin skin test response (TST) in 76 individuals of the Aché population. Poisson Regression was employed to assess risk genotypes. DRB1*04, DQA1*03 and DQB1*03:02 were associated with TST response in this population.

Cytokine gene polymorphisms are associated with susceptibility to tuberculosis in an Amerindian population.

SETTING: Cytokines play an important role in anti-tuberculosis immune response, combined with antigen-presenting cells and lymphocytes. Immune response gene polymorphisms have been reported to be associated with tuberculosis (TB) susceptibility in some but not all studies. OBJECTIVE: To evaluate the association of immune response genes with susceptibility to tuberculin skin test (TST) reactivity and/or TB. DESIGN: Fourteen single nucleotide polymorphisms were genotyped in 96 individuals of the Aché, a native Paraguayan population, by allelic discrimination using real-time polymerase chain reaction. Univariate and multivariate Poisson regression were employed to assess risk genotypes. RESULTS: A higher prevalence of purified protein derivative reactivity was associated with the TNF-α CCA/TCG haplotype (PR 1.298, 95%CI 1.059-1.589) and with the IL-10 AT/CC diplotype (PR 1.181, 95% CI 1.024-1.362), and the presence of the IL-8 rs4073 T allele was associated with protection against TB (PR 0.482, 95%CI 0.273-0.851). CONCLUSIONS: These results suggest that polymorphisms in genes associated with immune response are involved in TST reactivity and susceptibility to TB in the Aché population.

Distribution patterns of variability for 18 immune system genes in Amerindians--relationship with history and epidemiology.

Native American populations generally have a higher prevalence of infectious diseases than non-Native populations and this fact can induce different pressures in their immune system. We investigated the patterns of population differentiation (FST ) of 32 polymorphisms related to adaptive immune response in four Native American populations (Aché, Guarani-Kaiowá, Guarani-Ñandeva and Kaingang), and the results were compared with the three major world population data [Yoruba of Ibadan, Nigeria (YRI), Utah residents with northern and Western Europe ancestry (CEU) and Han Chinese of Beijing, China (CHB)] available in the HapMap database. The Aché clearly differentiated from the other Amerindians, but when all Native Americans were compared with the samples of other ethnic groups the lowest difference (0.08) was found with CHB (Asians), the second lowest (0.15) with YRI (Africans) and the most marked with CEU (European-derived). The considerable intra and interethnic differences found can be explained both in terms of diverse evolutionary distances and more recent environmental pathogen exposures; and they should be appropriately considered prior to any specific public health action.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Astrocytes induce manganese superoxide dismutase in brain capillary endothelial cells.

Astrocytes induce blood-brain barrier (BBB) properties in brain endothelial cells (EC)*O(2)*, generated in blood and EC, opens the BBB. Hence, high activity of superoxide dismutase (SOD) is a prerequisite for normal BBB function. Therefore, the influence of rat astrocytes on the expression of manganese (Mn)SOD in rat EC was investigated in two coculture models of the BBB, allowing either exchange of soluble factors or additionally cellular contacts. Activity, protein content and mRNA expression of endothelial MnSOD were significantly increased in both coculture models in comparison to monoculture by soluble astrocytic factors, such as cytokines. High activity of endothelial MnSOD may be considered as a further essential property of the BBB, which is induced and maintained by astrocytes.

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress.

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.

Glutathione, oxidative stress and neurodegeneration.

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species and mitochondrial dysfunction. Here, we review the evidence for a disturbance of glutathione homeostasis that may either lead to or result from oxidative stress in neurodegenerative disorders. Glutathione is an important intracellular antioxidant that protects against a variety of different antioxidant species. An important role for glutathione was proposed for the pathogenesis of Parkinson's disease, because a decrease in total glutathione concentrations in the substantia nigra has been observed in preclinical stages, at a time at which other biochemical changes are not yet detectable. Because glutathione does not cross the blood-brain barrier other treatment options to increase brain concentrations of glutathione including glutathione analogs, mimetics or precursors are discussed.

Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo.

MPTP produces clinical, biochemical, and neuropathologic changes reminiscent of those that occur in idiopathic Parkinson's disease (PD). In the present study we show that MPTP treatment led to activation of microglia in the substantia nigra pars compacta (SNpc), which was associated and colocalized with an increase in inducible nitric oxide synthase (iNOS) expression. In iNOS-deficient mice the increase of iNOS expression but not the activation of microglia was blocked. Dopaminergic SNpc neurons of iNOS-deficient mice were almost completely protected from MPTP toxicity in a chronic paradigm of MPTP toxicity. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites did not differ between iNOS-deficient mice and their wild-type littermates, this protection was not associated with a preservation of nigrostriatal terminals. Our results suggest that iNOS-derived nitric oxide produced in microglia plays an important role in the death of dopaminergic neurons but that other mechanisms contribute to the loss of dopaminergic terminals in MPTP neurotoxicity. We conclude that inhibition of iNOS may be a promising target for the treatment of PD.

Protection by synergistic effects of adenovirus-mediated X-chromosome-linked inhibitor of apoptosis and glial cell line-derived neurotrophic factor gene transfer in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, and neuropathological changes reminiscent of those occurring in idiopathic Parkinson's disease (PD). Here we show that a peptide caspase inhibitor, N-benzyloxy-carbonyl-val-ala-asp-fluoromethyl ketone, or adenoviral gene transfer (AdV) of a protein caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), prevent cell death of dopaminergic substantia nigra pars compacta (SNpc) neurons induced by MPTP or its active metabolite 1-methyl-4-phenylpyridinium in vitro and in vivo. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites does not differ between AdV-XIAP- and control vector-treated mice, this protection is not associated with a preservation of nigrostriatal terminals. In contrast, the combination of adenoviral gene transfer of XIAP and of the glial cell line-derived neurotrophic factor to the striatum provides synergistic effects, rescuing dopaminergic SNpc neurons from cell death and maintaining their nigrostriatal terminals. These data suggest that a combination of a caspase inhibitor, which blocks death, and a neurotrophic factor, which promotes the specific function of the rescued neurons, may be a promising strategy for the treatment of PD.

Cellular distribution of superoxide dismutases in the rat CNS.

Superoxide dismutase (SOD) is considered to be a major factor in protection of nervous tissue against excitotoxic and ischemic/hypoxic lesion. Controversial reports about the localization of SOD after such an insult prompted us to re-investigate immunocytochemically the localization of the enzyme in the brain and spinal cord using specific antibodies against the manganese (Mn-SOD) and copper/zinc (Cu/Zn-SOD) containing isoenzyme in combination with cell type specific markers. CNS tissue sections were analyzed by confocal laser scanning microscopy and digital photo imaging. Cu/Zn-SOD immunoreactivity was found to be located predominantly in astrocytes throughout the CNS. The staining was found in the cytoplasm, in cellular processes and, less intensive, in the nucleus sparing the nucleolus. At a lower level the enzyme was also detectable in neuronal perikarya and in structures of the neuropil. Motoneurons of the spinal cord displayed an enhanced Cu/Zn-SOD staining intensity, when compared to brain neurons. In contrast the Mn-containing isoenzyme was predominantly localized to neurons and their processes throughout the brain and the spinal cord. Confirming the mitochondrial localization of the enzyme, a granular staining pattern sparing the nucleus was observed. Mn-SOD stained mitochondria were also seen in astroglial cells but the staining intensity was, on the whole, much lower compared to neurons, and often hardly detectable. It seems reasonable to conclude that differences in the basal content of SOD-isoenzymes may contribute to different cellular susceptibilities in neurodegenerative processes that are accompanied by oxidative stress.

Enhanced cellular glutathione peroxidase immunoreactivity in activated astrocytes and in microglia during excitotoxin induced neurodegeneration.

The cellular distribution pattern of cellular glutathione peroxidase (GPx) was analyzed immunocytochemically in the normal rat central nervous system (CNS) and following exposure to the excitotoxin quinolinic acid (Quin). In the normal CNS, GPx was localized predominantly to microglia, identified by staining with isolectin B4 or the monoclonal antibody OX-42. Three days after intrastriatal administration of 90 pmoles Quin, GPx immunoreactivity was increased in activated microglia and also in astrocytes costained for glial fibrillary acidic protein (GFAP). Whereas GPx-positive astrocytes were seen predominantly in the environment of the lesion, most intensive immunoreactivity was located in globular-shaped microglia in the lesion core. GPx staining was generally low in neurons and failed to increase its intensity after lesion. In the case of excitotoxin-induced generation of oxygen-derived free radicals, the elevation of GPx levels in microglia, and likewise in activated astroglia, may provide an important mechanism to withstand oxidative stress.

Differential expression of superoxide dismutase isoforms in neuronal and glial compartments in the course of excitotoxically mediated neurodegeneration: relation to oxidative and nitrergic stress.

To examine the cellular distribution of radical scavenging enzymes in glia, in comparison to that in neurons and their behaviour during excitotoxically induced neurodegenerative processes, protein levels and the cellular localization of cytosolic and mitochondrial superoxide dismutase (Cu/Zn- and Mn-SOD) were investigated in the rat brain undergoing quinolinic acid (Quin)-induced neurodegeneration. Evidence for the specificity of the applied antibodies to detect immunocytochemically these SOD isoforms was obtained from electron microscopy and Western blotting. In control striatum Mn-SOD was clearly confined to neurons, whereas Cu/Zn-SOD was found, rather delicately, only in astrocytes. Microglia failed to stain with antibodies to both SOD isoforms. Quin application resulted in an initial formation of oxygen and nitrogen radicals as determined by the decline in the ratio of ascorbic to dehydroascorbic acid and by increased levels of nitrated proteins, an indicator for elevated peroxynitrite formation. Morphologically, massive neuronal damage was seen in parallel. Astroglia remained intact but showed initially decreased glutamine synthetase activities. The levels of Mn-SOD protein increased 2-fold 24 h after Quin injection (Western blotting) and declined only slowly over the time period considered (10 days). Cu/Zn-SOD levels increased only 1.3-fold. Immunocytochemical studies revealed that the increase in Mn-SOD is confined to neurons, whereas that of Cu/Zn-SOD was observed only in astroglial cells. Quiescent microglial cells were, as a rule, free of immunocytochemically detectable SOD, whereas in activated microglia a few Mn-SOD immunolabeled mitochondria occurred. Our results suggest a differential protective response in the Quin lesioned striatum in that Mn-SOD is upregulated in neurons and Cu/Zn-SOD in astroglia. Both SOD-isoforms are assumed to be induced to prevent oxidative and nitric oxide/peroxynitrite-mediated damage. In the border zone of the lesion core this strategy may contribute to resist the noxious stimulus.