Decreased expression of ectonucleotidase E-NPP1 in leukocytes from subjects with severe asthma exacerbation.

Several studies suggest that ATP and related nucleotides play a role in the pathophysiology of asthma. However, the functionality of ectonucleotidases in this disease has been scantly investigated. We studied total ectonucleotidase activity in leukocytes from patients suffering from asthma exacerbation and explored the expression of E-NTPDase 1, 2, 3, and 8, and E-NPP1, 2, and 3, in their polymorphonuclear cells by immunofluorescence and qPCR. Leukocytes from patients with mild or moderate asthma exacerbation had similar ectonucleotidase activity than leukocytes from healthy subjects, while in patients with severe asthma exacerbation, this activity was lower. Of the ectonucleotidases studied, only E-NPP1 displayed diminished immunofluorescence and a significant decrease in its mRNA expression, both in patients with severe asthma exacerbation. This reduced E-NPP1 expression could be responsible for increased amounts of ATP or other nucleotides, capable of worsening asthma exacerbation, and warranting further investigation.

Inhibition of extracellular nucleotides hydrolysis intensifies the allergic bronchospasm. A novel protective role of ectonucleotidases.

BACKGROUND: Nucleotides released to the extracellular space stimulate purinergic receptors, and their effects are modulated by ectonucleotidases. The role of ATP in the allergic bronchospasm has been scantly studied. METHODS: We used several techniques (plethysmography, organ baths, confocal microscopy, RT-PCR, ATP measurement) to explore the role of nucleotides and ectonucleotidases in the allergic bronchospasm in guinea pigs. RESULTS: While allergenic challenge with a low-dose ovalbumin (OVA) only produced a small bronchospasm (~2-fold the basal lung resistance), previous inhibition of ectonucleotidases by ARL-67156 greatly intensified this response (~11-fold the basal lung resistance, with 44% mortality). Bronchoalveolar lavage fluid obtained during this bronchospasm contained increased ATP concentration. This potentiation was abolished by antagonism of purinergic receptors (suramin+RB2) or TXA2 receptor (SQ29548), or by intratracheal apyrase. In tracheal rings and lung parenchyma strips, OVA caused a concentration-dependent contraction. Suramin+RB2 or levamisole produced a significant rightward displacement of this response, and ARL-67156 did not modify it. Platelets stimulated with OVA released ATP. Confocal images of nonsensitized tracheas showed slight fluorescence for P2Y6 receptors in epithelium and none for P2Y4 . Sensitized animals showed strong fluorescence to both receptors and to alkaline phosphatase in the airway epithelium. This correlated with a large increment in mRNA for P2Y4 and P2Y6 receptors in sensitized animals. CONCLUSIONS: Nucleotides greatly potentiate the allergic bronchospasm when ectonucleotidases activity is diminished, and this effect is probably favored by the upregulation of P2Y4 and P2Y6 receptors in airway epithelium during sensitization. These results prompt for further research on these mechanisms in human asthma.

Environmental sex determination mechanisms in reptiles.

Temperature-dependent sex determination (TSD) was first discovered in reptiles. Since then, a great diversity of sex-determining responses to temperature has been reported. Higher temperatures can produce either males or females, and the temperature ranges and lengths of exposure that influence TSD are remarkably variable among species. In addition, transitory gene regulatory networks leading to gonadal TSD have evolved. Although most genes involved in gonadal development are conserved in vertebrates, including TSD species, temporal and spatial gene expression patterns vary among species. Despite variation in TSD pattern and gene expression heterochrony, the structural framework, the medullary cords, and cortex of the bipotential gonad have been strongly conserved. Aromatase (CYP19), which regulates gonadal estrogen levels, is proposed to be the main target of a putative thermosensitive factor for TSD. However, manipulation of estrogen levels rarely mimics the precise timing of temperature effects on expression of gonadal genes, as occurs with TSD. Estrogen levels may influence sex determination or gonad differentiation depending on the species. Furthermore, the process leading to sex determination under the influence of temperature poses problems that are not encountered by species with genetic sex determination. Yolk steroids of maternal origin and steroids produced by the embryonic nervous system should also be considered as sources of hormones that may play a role in TSD.

Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination.

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination.

Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation.

Sex determination in mammalian gonads depends on the concerted action of SRY and SOX at the genital ridge. Although most research on the mechanisms involved in sex determination has been done in mice, the study of non-model organisms may indicate the extent of the generalizations currently based on model systems. The present study investigated the correlation between SRY/SOX9 expression patterns and the process of morphogenesis in the developing gonad of the rabbit. In males, the onset of SRY/SOX9 expression closely followed the establishment of undifferentiated genital ridges at 13 to 14 dpc. In contrast to mouse, real-time PCR in the rabbit revealed that levels of SRY/ SOX9 peak after the onset of seminiferous cord formation, while in mouse this occurs before. Furthermore, rabbit gonads maintain low levels of SRY and SOX9 expression in male and female gonads, respectively. In situ hybridization suggests that cells of the mesonephric Bowman capsules, which do not express SRY, may become SOX9-expressing pre-Sertoli cells during the long period of seminiferous cord formation in the rabbit. In contrast to mouse, current results indicate that the patterns of SRY/SOX9 expression associated with the process of gonadal morphogenesis in rabbit appear similar to those of other mammals, including humans.