Expression of mammalian RF-amide peptides neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the PrRP receptor in the peripheral tissues of the rat

The mRNA expression of neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the UHR-1/GPR10 receptor were examined using in situ hybridization in rat peripheral tissues. In the hypophysis, modest expression of PrRP and receptor mRNA were seen in the anterior lobe. The trigeminal ganglion was devoid of expression signals. PrRP and UHR-1/GPR10 receptor mRNA:s were found in the adrenal medulla and PrRP mRNA was found in the pancreas. NPFF mRNA was detected in the spleen. In the testis and epididymis, PrRP and UHR-1/GPR10 receptor mRNA:s were detected. The results suggest a limited expression of mammalian RF-amide peptides in the peripheral organs.     

Stimulatory and inhibitory action of cytokines on the regulation of hCMV-IE promoter activity in human endothelial cells

Viral promoters are commonly used as regulatory elements in gene therapy vectors due to their strong activity in various cell lines in vitro. However, transgene expression under the control of viral promoters in vivo has been shown to be limited to a short period of time. Several mechanisms for the transient expression of the delivered transgene may be important including deletion of transduced cells or promoter downregulation. Recently we reported that cytokines may either decrease or increase the activity of the human cytomegalovirus (hCMV) promoter in monocytes depending on the differentiation status of the transduced cells. For many applications, the gene of interest has to be delivered into an inflammatory milieu (tumour, ischaemia/reperfusion, vector-induced inflammation etc.). In this report we investigated the influence of various inflammatory cytokines on the hCMV-IE promoter activity in transduced human primary endothelial cells (Huvec) in vitro, which may be the first target cells after gene transfer into different organs. Cultured cells were infected with an E1-deleted adenoviral vector encoding for E. colibeta-galactosidase (Adbeta-gal) driven by the hCMV-IE promoter and incubated either with or without various cytokines. Our results indicate that interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) downregulate promoter activity in endothelial cells whereas, in contrast, tumour necrosis factor (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 4 (IL-4) increased the promoter activity. These results suggest that inflammatory processes influence the in vivo expression of transferred viral promoter controlled genes of interest.     

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.     

Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection

Notch1 is involved in directing cell fate decisions in a variety of developmental scenarios. Extending previous experiments in mice, we generated transgenic rats expressing the intracellular domain of Notch1 in the thymus. Importantly, this leads to sustained expression of the pre-TCR throughout thymocyte development, accompanied by a reduction of alphabetaTCR complexes. In addition, re-expression of RAG-1 and RAG-2 in TCRbeta(+) cells is impaired, and the Valpha repertoire is altered. Consequently, thymocytes in transgenic rats do not undergo positive selection and largely fail to progress to the single positive stage. According to our model, the previously reported effects of Notch1 on the CD4/CD8 cell fate decision may be explained by a differential sensitivity of the two lineages toward altered TCR signaling.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

Species-specific Effects of Vascular Plants on Carbon Turnover and Methane Emissions from Wetlands

Species composition affects the carbon turnover and the formation and emission of the greenhouse gas methane (CH₄) in wetlands. Here we investigate the individual effects of vascular plant species on the carbon cycling in a wetland ecosystem. We used a novel combination of laboratory methods and controlled environment facilities and studied three different vascular plant species (Eriophorum vaginatum, Carex rostrata and Juncus effusus) collected from the same wetland in southern Sweden. We found distinct differences in the functioning of these wetland sedges in terms of their effects on carbon dioxide (CO₂) and CH₄ fluxes, bubble emission of CH₄, decomposition of ¹⁴C-labelled acetate into ¹⁴CH₄ and ¹⁴CO₂, rhizospheric oxidation of CH₄ to CO₂ and stimulation of methanogenesis through root exudation of substrate (e.g., acetate). The results show that the emission of CH₄ from peat–plant monoliths was highest when the vegetation was dominated by Carex (6.76 mg CH₄ m⁻² h⁻¹) than when it was dominated by Eriophorum (2.38 mg CH₄ m⁻² h⁻¹) or Juncus (2.68 mg CH₄ m⁻² h⁻¹). Furthermore, the CH₄ emission seemed controlled primarily by the degree of rhizospheric CH₄ oxidation which was between 20 and 40% for Carex but >90% for both the other species. Our results point toward a direct and very important linkage between the plant species composition and the functioning of wetland ecosystems and indicate that changes in the species composition may alter important processes relating to controls of and interactions between greenhouse gas fluxes with significant implications for feedback mechanisms in a changing climate as a result.     

Functional significance of responsiveness to capacitating conditions in boar spermatozoa

New methods are needed for rapid and sensitive assessment of sperm function. As the ability to fertilize an oocyte is acquired during the capacitation process, assessments of sperm function have to be performed under fertilizing conditions. In this study, we monitored the dynamics of the temporal response of sperm from ejaculates of both fertile and subfertile boars to capacitating conditions in vitro (responsiveness) by following the changes in the response to calcium ionophore treatment and in [Ca(2+)](i). The differences between individual males were also investigated. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were found to progress as a function of time during incubation under capacitating conditions. After primary kinetic analysis, 120 min was chosen as the point in time for assessment of responsiveness. Intra-boar variability in responsiveness parameters was relatively high (variation coefficient CV>30%), especially in the response to ionophore treatment, indicating that an isolated test may be inadequate for the evaluation of sperm function. Despite this high variability, there were markedly significant individual differences with respect to changes during capacitation, and there were significant correlations between conventional and responsiveness sperm parameters. The population of samples from subfertile boars, was found to be heterogeneous in regard to sperm responsiveness to capacitating conditions. There were two significantly different classes of subfertile boars ("low" and "high" responders), indicating that fertility may be associated with suboptimal rather than maximal response (both too rapid and too slow membrane changes). Therefore, criteria for quality judgement should include both the low and upper limits of responsiveness. The use of responsiveness parameters together with conventional spermatological parameters improved the prediction level of multiple regression models for farrowing rate and litter size. It can be concluded that the combination of sperm responsiveness parameters applied here is a suitable tool for the evaluation of sperm function.