A model for preservation of thymocyte-depleted thymus.

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

A model for preservation of thymocyte-depleted thymus

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Effects of aerobic exercise on molecular aspects of asthma: involvement of SOCS-JAK-STAT.

BACKGROUND: Aerobic training (AT) decreases airway inflammation in asthma, but the underlying cellular and molecular mechanisms are not completely understood. Thus, this study evaluated the participation of SOCS-JAK-STAT signaling in the effects of AT on airway inflammation, remodeling and hyperresponsiveness in a model of allergic airway inflammation. METHODS: C57Bl/6 mice were divided into Control (Co), Exercise (Ex), HDM (HDM), and HDM+Exercise (HDM+ Ex). Dermatophagoides pteronyssinus (100ug/mouse) were administered oro-tracheally on days 0, 7, 14, 21, 28, 35, 42 and 49. AT was performed in a treadmill during 4 weeks in moderate intensity, from day 24 until day 52. RESULTS: AT inhibited HDM-induced total cells (p<0.001), eosinophils (p<0.01), neutrophils (p<0.01) and lymphocytes (p<0.01) in BAL, and eosinophils (p<0.01), neutrophils (p<0.01) and lymphocytes (p<0.01) in peribronchial space. AT also reduced BAL levels of IL-4 (p<0.001), IL-5 (p<0.001), IL-13 (p<0.001), CXCL1 (p<0.01), IL-17 (p<0.01), IL-23 (p<0.05), IL-33 (p<0.05), while increased IL- 10 (p<0.05). Airway collagen fibers (p<0.01), elastic fibers p<0.01) and mucin (p<0.01) were also reduced by AT. AT also inhibited HDM-induced airway hyperresponsiveness (AHR) to methacholine 6,25mg/ml (p<0.01), 12,5mg/mL (p<0.01), 25mg/mL (p<0.01) and 50mg/mL (p<0.01). Mechanistically, AT reduced the expression of STAT6 (p<0.05), STAT3 (p<0.001), STAT5 (p<0.01) and JAK2 (p<0.001), similarly by peribronchial leukocytes and by airway epithelial cells. SOCS1 expression (p<0.001) was upregulated in leukocytes and in epithelial cells, SOCS2 (p<0.01) was upregulated in leukocytes and SOCS3 down-regulated in leukocytes (p<0.05) and in epithelial cells (p<0.001). CONCLUSIONS: AT reduces asthma phenotype involving SOCSJAK- STAT signaling.

Honey protects against wings posture error and molecular changes related to mitochondrial pathways induced by hypoxia/reoxygenation in adult Drosophila melanogaster.

We conducted an investigation to evaluate the effects of Brazilian Pampa biome honey and its major phenolic compounds on the development of an erected wings posture phenotype and related mitochondrial aspects induced by Hypoxia/Reoxygenation (H/R) in Drosophila melanogaster. Flies were pre-treated for 3 days with a 10% honey solution and different concentrations of caffeic acid and ρ-coumaric acid and then submitted to hypoxia for 3 h. We observed that after reoxygenation, some flies acquired an erected wings posture and that this feature may be related to mortality. In addition, H/R induced down-regulation of ewg mRNA expression, which could be associated to the observed complex phenotype. H/R also caused a dysregulation in opa1-like, ldh and diap genes expression and reduced O2 fluxes in flie's mitochondria. Honey mitigated opa1-like mRNA expression changes provoked by H/R. Differently from honey, caffeic and ρ-coumaric acids displayed no protective effects. In conclusion, we report for the first time the protective effects of honey against complex phenotypes and mitochondrial changes induced by H/R in adult flies.

Data on the phosphorylation of p38MAPK and JNK induced by chlorpyrifos in Drosophila melanogaster.

Exposure to organophosphate compounds, such as chlorpyrifos, has been linked to disturbances on cell signaling pathways. Mitogen activated protein kinases (MAPK) are a family of protein kinases involved in a range of cellular processes, including stress response, apoptosis and survival. Therefore, changes in the activation state of these kinases may characterize key mechanisms of toxicity elicited by xenobiotics. Here we report data on the phosphorylation of p38MAPK and JNK, members of the MAPK family, in Drosophila melanogaster exposed to chlorpyrifos, as characterized by western blotting assays.

Carbon disulfide formation in papaya under conditions of dithiocarbamate residue analysis.

Golden, Sunrise Solo and Tainung cultivars of papaya were found to release CS2 when submitted to experimental conditions of dithiocarbamate residue analysis. Three common analytical methods were used to quantitate CS2; one spectrophotometric method and two chromatographic methods. All three methods gave positive CS2 results for all three papaya varieties. Other endogenous compounds present in isooctane extracts of papaya fractions detected via gas chromatography (GC/ITD) using electron ionization (EI) were: carbonyl sulfide, dimethyl sulfide, carbon disulfide, 2-methylthiophene, 3-methylthiophene, 2-ethylthiophene, 3-ethylthiophene, benzylisothiocyanate, benzylthiocyanate and benzonitrile. Control samples were obtained from papaya plantations cultivated in experimental areas, in which no treatment with fungicides of the dithiocarbamate group was applied. Endogenous CS2 levels were compared with true dithiocarbamate residues measured in papaya samples from the field trials following applications of the mancozeb fungicide. Three days after application, true dithiocarbamate residues, measured by the procedure with isooctane partitioning and GC-ITD, were at the average level of 2 mg kg(-1).

Assessment of water pollution in the Brazilian Pampa biome by means of stress biomarkers in tadpoles of the leaf frog Phyllomedusa iheringii (Anura: Hylidae).

The Brazilian Pampa biome is currently under constant threat due to increase of agriculture and improper management of urban effluents. Studies with a focus on the assessment of impacts caused by human activities in this biome are scarce. In the present study, we measured stress-related biomarkers in tadpoles of the leaf frog Phyllomedusa iheringii, an endemic species to the Pampa biome, and tested its suitability as a bioindicator for the assessment of potential aquatic contamination in selected ponds (S1 and S2) nearby agricultural areas in comparison to a reference site. A significant decrease in acetylcholinesterase activity was observed in S2 when compared to S1 and reference. The levels of total-hydroperoxides were increased in S2 site. In parallel, increased activity of the antioxidant enzymes catalase, superoxide dismutase and glutathione S-transferase were observed in S2 when compared to S1 and reference. Further studies are necessary in order to correlate the changes observed here with different chemical stressors in water, as well as to elucidate mechanisms of toxicity induced by pesticides in amphibian species endemic to the Pampa biome. Nevertheless, our study validates Phyllomedusa iheringii as a valuable bioindicator in environmental studies.

Oxidative stress markers in fish (Astyanax sp. and Danio rerio) exposed to urban and agricultural effluents in the Brazilian Pampa biome.

Aquatic ecosystems are under constant risk due to industrial, agricultural, and urban activities, compromising water quality and preservation of aquatic biota. The assessment of toxicological impacts caused by pollutants to aquatic environment using biomarker measurements in fish can provide reliable data to estimate sublethal effects posed by chemicals in contaminated areas. In this study, fish (Astyanax sp. and Danio rerio) exposed to agricultural and urban effluents at the Vacacaí River, Brazil, were tested for potential signs of aquatic contamination. This river comprehends one of the main watercourses of the Brazilian Pampa, a biome with a large biodiversity that has been neglected in terms of environmental and social-economic development. Sites S1 and S2 were chosen by their proximity to crops and wastewater discharge points, while reference site was located upstream of S1 and S2, in an apparently non-degraded area. Fish muscle and brain tissues were processed for determination of acetylcholinesterase as well as oxidative stress-related biomarkers. The results showed signs of environmental contamination, hallmarked by significant changes in cholinesterase activity, expression of metallothionein, antioxidant enzymes, glutathione levels, and activation of antioxidant/cell stress response signaling pathways in fish exposed to contaminated sites when compared to reference. Based on these results, it is evidenced that urban and agricultural activities are posing risk to the environmental quality of water resources at the studied area. It is also demonstrated that cell stress biomarkers may serve as important tools for biomonitoring and development of risk assessment protocols in the Pampa biome.

Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress?

Organic and inorganic forms of mercury are highly neurotoxic environmental contaminants. The exact mechanisms involved in mercury neurotoxicity are still unclear. Oxidative stress appears to play central role in this process. In this study, we aimed to validate an insect-based model for the investigation of oxidative stress during mercury poisoning of lobster cockroach Nauphoeta cinerea. The advantages of using insects in basic toxicological studies include the easier handling, rapid proliferation/growing and absence of ethical issues, comparing to rodent-based models. Insects received solutions of HgCl2 (10, 20 and 40mgL(-1) in drinking water) for 7d. 24h after mercury exposure, animals were euthanized and head tissue samples were prepared for oxidative stress related biochemical determinations. Mercury exposure caused a concentration dependent decrease in survival rate. Cholinesterase activity was unchanged. Catalase activity was substantially impaired after mercury treatment 40mgL(-1). Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20mgL(-1) and 40mgL(-1) comparing to control. These results were accompanied by decreased GSH levels and increased hydroperoxide and TBARS formation. In conclusion, our results show that mercuric compounds are able to induce oxidative stress signs in insect by modulating survival rate as well as inducing impairments on important antioxidant systems. In addition, our data demonstrates for the first time that Nauphoeta cinerea represents an interesting animal model to investigate mercury toxicity and indicates that the GSH and thioredoxin antioxidant systems plays central role in Hg induced toxicity in insects.

Structural alterations in adult rat carotid bodies exposed to hyperbaric oxygenation.

UNLABELLED: Inhibition of carotid body (CB) function is the main mechanism involved in the attenuation of respiratory drive observed during hyperoxia. However, only a few studies at 5.0 atmospheres absolutes (ATA) have analyzed carotid body structure or function in hyperbaric oxygenation (HBO2) situations. We hypothesized that rats will present CB structural alterations when exposed to different lower hyperbaric oxygen doses enough to alter their chemosensory response to hypoxia. METHODS: Twenty-one adult male Wistar rats, divided into three groups, were maintained in room air or exposed to O2 at 2.4 or 3.0 ATA for six hours. Histological, ultrastructural and immunohistochemical analyses for neuronal nitric oxide synthase (nNOS) and F2-isoprostane were performed in the excised CBs. RESULTS: Histological analyses revealed signs of intracellular edema in animals exposed to both conditions, but this was more marked in the 3.0 ATA group, which showed ultrastructural alterations at the mitochondrial level. There was a significant increase in the volume density of intraglomic-congested capillaries in the 3.0 ATA group associated with an arteriolar vasoconstriction. In the 2.4 ATA group, there was a relative increase of glomic light cells and a decrease of glomic progenitor cells. Additionally, there was a stronger immunoreactivity for F2-isoprostane in the 3.0 ATA O2-exposed carotid bodies. The glomic cells stained positive for nNOS, but no difference was observed between the groups. Our results show that high O2 exposures may induce structural alterations in glomic cells with signs of lipid peroxidation. We further suggest that deviation of blood flow toward intraglomic capillaries occurs in hyperbaric hyperoxia.

Baroreflex deficit blunts exercise training-induced cardiovascular and autonomic adaptations in hypertensive rats.

1. Baroreceptors regulate moment-to-moment blood pressure (BP) variations, but their long-term effect on the cardiovascular system remains unclear. Baroreceptor deficit accompanying hypertension contributes to increased BP variability (BPV) and sympathetic activity, whereas exercise training has been associated with an improvement in these baroreflex-mediated changes. The aim of the present study was to evaluate the autonomic, haemodynamic and cardiac morphofunctional effects of long-term sinoaortic baroreceptor denervation (SAD) in trained and sedentary spontaneously hypertensive rats (SHR). 2. Rats were subjected to SAD or sham surgery and were then further divided into sedentary and trained groups. Exercise training was performed on a treadmill (five times per week, 50-70% maximal running speed). All groups were studied after 10 weeks. 3. Sinoaortic baroreceptor denervation in SHR had no effect on basal heart rate (HR) or BP, but did augment BPV, impairing the cardiac function associated with increased cardiac hypertrophy and collagen deposition. Exercise training reduced BP and HR, re-established baroreflex sensitivity and improved both HR variability and BPV. However, SAD in trained SHR blunted all these improvements. Moreover, the systolic and diastolic hypertensive dysfunction, reduced left ventricular chamber diameter and increased cardiac collagen deposition seen in SHR were improved after the training protocol. These benefits were attenuated in trained SAD SHR. 4. In conclusion, the present study has demonstrated that the arterial baroreflex mediates cardiac disturbances associated with hypertension and is crucial for the beneficial cardiovascular morphofunctional and autonomic adaptations induced by chronic exercise in hypertension.

Early short-term versus prolonged low-dose methylprednisolone therapy in acute lung injury.

The present study compared the effects of early short-term with prolonged low-dose corticosteroid therapy in acute lung injury (ALI). In total, 120 BALB/c mice were randomly divided into five groups. In the control group, saline was intratracheally (i.t.) instilled. In the ALI group, mice received Escherichia coli lipopolysaccharide (10 microg i.t.). ALI animals were further randomised into four subgroups to receive saline (0.1 mL i.v.) or methylprednisolone (2 mg x kg(-1) i.v.) at 6 h, 24 h or daily (for 7 days, beginning at day 1). At 1, 3 and 8 weeks, in vivo and in vitro lung mechanics and histology (light and electron microscopy), collagen and elastic fibre content, cytokines in bronchoalveolar lavage fluid and the expression of matrix metalloproteinase (MMP)-9 and -2 were measured. In vivo (static elastance and viscoelastic pressure) and in vitro (tissue elastance and resistance) lung mechanics, alveolar collapse, cell infiltration, collagen and elastic fibre content and the expression of MMP-9 and MMP-2 were increased in ALI at 1 week. Methylprednisolone led to a complete resolution of lung mechanics, avoided fibroelastogenesis and the increase in the expression of MMP-9 and MMP-2 independent of steroid treatment design. Thus, early short-term, low-dose methylprednisolone is as effective as prolonged therapy in acute lung injury.

A simple method for the differential characterization of alveoli and alveolar ducts in injured lungs.

RATIONALE AND HYPOTHESIS: Previous studies evaluating the histoarchitecture of distal airspaces have been shown to be limited by the difficulty in adequately differentiating alveoli and alveolar ducts. This limitation has been specially noticed in studies addressing lung recruitment and in situations of diffuse alveolar damage (DAD), where generic nominations for distal airspaces had to be created, such as "peripheral airspaces" (PAS) and "large-volume gas-exchanging airspaces" (LVGEA). Elastic stains have been largely used to describe normal lung structures. Weigert's resorcin-fuchsin staining (WRF) demarcates the thickened free portions of the ductal septum facilitating its recognition. We hypothesized that this staining could help in differentiating alveoli from alveolar ducts in distorted lung parenchyma. MATERIAL AND METHODS: Samples of control lungs and of DAD lungs induced by mechanical ventilation (VILI) were stained with hematoxylin-eosin (HE) and with WRF. Using morphometry we assessed the volume proportion of alveoli, alveolar ducts and LVGEA in control and VILI lungs. RESULTS: WRF stained VILI lungs showed a significant decrease in the volume proportion of LVGEA and alveoli and a significant increase in the volume proportion of alveolar ducts when compared to HE stained samples. CONCLUSION: We conclude that WRF staining is useful to distinguish alveolar ducts from alveoli in a DAD model, and suggest that it should be routinely used when morphometric studies of lung parenchyma are performed.

Characterization of Ngef, a novel member of the Dbl family of genes expressed predominantly in the caudate nucleus.

We have identified Ngef as a novel member of the family of Dbl genes. Many members of this family have been shown to function as guanine nucleotide exchange factors for the Rho-type GTPases. Ngef is predominantly expressed in brain, with the strongest signal in the caudate nucleus, a region associated with the control of movement. Ngef contains a translated trinucleotide repeat, a polyglutamic acid stretch interrupted by a glycine. We have localized the Ngef gene to mouse chromosome 1 and the human homologue of Ngef to human chromosome 2q37. We have shown in preliminary experiments that Ngef has transforming potential in cell culture and is able to induce tumors in nude mice.

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5' end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3' to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Analysis of mutations in the tudor domain of the survival motor neuron protein SMN.

Autosomal recessive childhood onset spinal muscular atrophy (SMA) is a leading cause of infant mortality caused by mutations in the survival motor neuron (SMN) gene. The SMN protein is involved in RNA processing and is localised in structures called GEMs in the nucleus. Nothing is yet understood about why mutations in SMN gene result in the selective motor neuron loss observed in patients. The SMN protein domains conserved across several species may indicate functionally significant regions. Exon 3 of SMN contains homology to a tudor domain, where a Type I SMA patient has been reported to harbour a missense mutation. We have generated missense mutants in this region of SMN and have tested their ability to form GEMs when transfected into HeLa cells. Our results show such mutant SMN proteins still localise to GEMs. Furthermore, exon 7 deleted SMN protein appears to exert a dominant negative effect on localisation of endogenous SMN protein. However, exon 3 mutant protein and exon 5 deleted protein exert no such effect.

Correlation of SMNt and SMNc gene copy number with age of onset and survival in spinal muscular atrophy.

Childhood-onset autosomal recessive spinal muscular atrophy (SMA) is associated with absence of the telomeric survival motor neuron gene (SMNt) in most patients, and deletion of the neuronal apoptosis inhibitory protein (NAIP) gene in the majority of severely affected patients. Analysis of SMNt has been complicated by the existence of a centromeric copy, SMNc, which is almost identical to SMNt but which can be distinguished from it by restriction enzyme analysis. In this study 143 SMA patients have been genotyped for the presence or absence of the SMNt, SMNc and NAIP genes, and the data correlated with quantifiable clinical variables. Although a significant correlation was observed between the presence or absence of the NAIP gene and the severity of the clinical phenotype in SMA patients generally, there was no difference in age of onset or survival in type I patients with the NAIP+ or NAIP- genotype. Fluorimetric PCR analysis of SMNc gene dosage in 57 patients homozygous for the absence of the SMNt gene but in whom the NAIP gene was present showed a highly significant correlation between SMNc copy number and SMA subtype, and between SMNc copy number and both age of onset and length of survival. The data provide strong statistical support for the emerging consensus that the clinical phenotype in SMA is directed primarily by the level of functional SMN protein. The lower SMNc copy number in type I patients in whom the NAIP gene is present suggests that the SMNt gene is removed by deletion in the majority of such patients, rather than by gene conversion as is the case in SMA types II and III.

Genomic organization and chromosomal localization of a member of the MAP kinase phosphatase gene family to human chromosome 11p15.5 and a pseudogene to 10q11.2.

Mitogen-activated protein kinase phosphatases (MKPs) play a central role in a variety of signaling pathways. We recently described a novel murine MKP, M3/6, which is uniquely specific for c-Jun N-terminal kinase/stress-activated protein kinase and p38 kinase. Here we report the localization of the human orthologue of this gene, HB5, to within 150 kb of H19 on human chromosome 11p15.5. The gene consists of six exons. Two of the introns in HB5 are not found in other genes of this family, suggesting an evolutionary split between MKPs displaying specificity toward different MAP kinases. An intronless pseudogene is present on chromosome 10q11.2. Although 11p15.5 is an imprinted region, HB5 is almost entirely unmethylated on both alleles in lymphocytes. Chromosome 11p15 has been implicated in the development of a number of tumor types, including lung, a tissue known to express this gene. Loss of heterozygosity was found in one of eight informative lung tumors studied.

Gene conversion at the SMN locus in autosomal recessive spinal muscular atrophy does not predict a mild phenotype.

Autosomal recessive proximal spinal muscular atrophy (SMA) is a disease of motor neuron death and a common cause of morbidity in childhood. It has been mapped to 5q13 and shown to be associated with deletions in a gene which has been called the survival motor neuron (SMN) gene. SMN exists in two copies in 5q13 and deletions in exon 7 and 8 of the telomeric copy (SMNtel) occur in over 90% of patients regardless of disease severity. In contrast, deletion of exon 7 and 8 of the centromeric copy of SMN is present in 3-5% of the normal population. In a minority of patients, exon 7 but not exon 8 of SMNtel appears deleted. The purpose of this study was to analyse this latter type of deletion in more detail. In all patients where there was absence of PCR amplification of exon 7 but not exon 8 of SMNtel this was found to be due to replacement with the homologous copy of SMNcen by a possible gene conversion event. This type of mutation occurred in all grades of severity of SMA.

[Spinal cord injuries in children]. / Traumatismo raquimedular em crianças.

Spinal cord injuries are rare in children, in face of their higher mobility comparing to adults. The high cervical and the thoracic segments of the spine are more frequently affected. In the last 10 years we had 90 cases of spinal injuries in our service being 12 with neurologic deficient (8 male and 4 female) and four of them without radiographic abnormality, even in the dynamics studies. The authors emphasise the possibility of occurrence of neurologic deficit in children after trauma, even without any radiographic abnormality.