BackgroundFractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain.ObjectiveTo investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma.MethodsFractional exhaled nitric oxide was measured in healthy controls (n = 14) and allergic asthmatics (n = 12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis.ResultsFractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression.ConclusionHigher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene Sry, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named testatin. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that testatin expression is restricted to fetal gonads and adult testis. Furthermore, testatin is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of Sry action, at a time immediately after the peak of Sry expression. This finding suggests that testatin might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.

In order to study estrogen effects on developing human neurons, we have established primary cultures of neurons and glia from 8-13-week human embryo cortex and spinal cord. The neuronal identity of the cultures was verified using the neuronal synaptic vesicle and neuronal endosomal membrane markers synaptotagmin, synapsin and synaptophysin, and the glial contribution to the mixed glial-neuronal cultures was verified using the glial marker glial fibrillary acidic protein (GFAP). We here report expression of estrogen receptor beta (ERbeta) in these cells using RT-PCR and sequencing, RNAse protection assay, immunohistochemistry and immunoblotting. We found that both neuronal and mixed glial-neuronal cultures expressed ERbeta. Treatment with 17beta-estradiol gave an increased expression of ERbeta in both types of cultures. These results suggest that ERbeta is expressed in fetal brain and thus may mediate effects of estrogen in the developing nervous system. Furthermore, the results suggest that expression of ERbeta in fetal brain may be regulated by estrogen.

Recent studies have strongly indicated that at least three regions [azoospermia factor (AZF) a-c] on the long arm of the Y-chromosome code for factors involved in spermatogenesis. In order to reveal the prevalence of microdeletions in these regions in a Swedish population, 192 men consecutively referred to our andrology unit due to infertility and showing oligozoospermia (n=53) or azoospermia (n=139) but no obstruction or hormonal disturbances, were investigated. For this study we used a multiplex polymerase chain reaction (PCR) method including 13 pairs of primers divided into five different primer mixes. It was found that four men, all with azoospermia, had deletions including part of the AZFb region and probably the entire AZFc region. Testis biopsies showed different morphology ranging from absence of germ cells to hypospermatogenisis. Of special interest was one patient that was first investigated 10 years ago due to primary infertility and oligozoospermia. Today he has developed azoospermia. It is concluded that the number of patients with microdeletions on the Y chromosome is rather low (less than 3% in highly selected azoospermic men) in our study compared to a number of other studies in which a 1-55% incidence have been reported. It is possible that ethnic differences, selection criteria and methodological aspects can contribute to the difference between the present and previous studies.

We have recently found that values of the transforming growth factor (TGF)β1 in human ovarian follicular fluid obtained during ovarian stimulation for IVF were higher in women who subsequently became pregnant following embryo transfer. We therefore postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and improve the chances of a successful implantation. We have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the presence in human oocytes and preimplantation embryos of the essential components of the TGFβ signalling pathway, TGFβ receptors type I and II and the substrate proteins Smad 2 and 3. We found that both receptors, as well as Smad 2 and 3, were present in the unfertilized oocyte, whereas only the type I receptor and Smad 2 and 3 were present at the blastocyst stage. At the 4-cell and 8-cell stages neither of the receptors was present, but Smad 2 and 3 were present at both stages. These findings support our hypothesis that the TGFβ1 in follicular fluid may interact with the oocyte and preimplantation embryo via TGFβ receptors, and that TGFβ signalling may be important for the development of the oocyte and the preimplantation embryo.