Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.     

Tissue banking training courses: Polish experience

Personnel directly involved in the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells should be appropriately qualified and provided with timely and relevant training according to EU directives. In the time of new tissue and cells regulations implementation such a training system existed in Poland only at a local level. The first training programme outlines for various groups of health professionals engaged in tissue banking practice was created in co-operation with the Institute for LifeLong Learning at University of Barcelona in 2006. This initial training courses were financially supported by EU Transition Facility Programme 2004. Then, starting from 2006, based on previous experience, system of advanced training courses was created. This training programme was financially supported by the National Programme for the Development of Transplantation Medicine 2006–2009—POLGRAFT financed by Polish Ministry of Health. During 2006 and 2007 first set of tissue banking initial training courses were provided according to TF 2004 project. Over 200 pathologists, forensic medicine specialists and other medical doctors responsible for donor screening and classification, medical directors of tissue establishments, technical staff; tissue graft users: orthopaedic surgeons, neurosurgeons, cardiosurgeons and ophthalmologists were trained. Between 2006 and 2009 there were organized 8 advanced tissue banking training courses according to POLGRAFT programme. There were organized both theoretical and practical courses on various aspects of tissue for over 350 persons. We present our experience in organisation of international and national tissue banking training courses.     

The influence of adiponectin on the transcriptomic profile of porcine luteal cells

Reproductive functions are closely related to nutritional status. Recent studies suggest that adiponectin may be a hormonal link between them. Adiponectin is an adipocytokine, abundantly expressed in adipose tissues. It plays a dominant role in lipid and carbohydrate metabolism by stimulating fatty acid oxidation, decreasing plasma triglycerides, and increasing cells’ sensitivity to insulin and has direct antiatherosclerotic effects. The hormone is also postulated to play a modulatory role in the regulation of the reproductive system. The aim of this study was to identify differentially expressed genes (DE-genes) in response to adiponectin treatment of porcine luteal ovarian cells. The global expression of genes in the porcine ovary was investigated using the Porcine (V2) Two-color gene expression microarray, 4?×?44 (Agilent, USA). Analysis of the microarray data showed that 701 genes were differentially expressed and 389 genes showed a fold change greater than 1.2 (p?<?0.05). Among this number, 186 genes were up-regulated and 203 were down-regulated. The list of DE-genes was used for gene ontology analyses. The biological process list was generated from up-regulated and down-regulated DE-genes. We found that up-regulated products of DE-genes take part in 30 biological processes and down-regulated products in 9. Analysis of the interaction network among DE-genes showed that adiponectin interacts with genes involved in important processes in luteal cells. These results provide a basis for future work describing the detailed interactions and relationships explaining local regulation of adiponectin actions in the ovary of pigs.     

Serum Metabolomic Response of Myasthenia Gravis Patients to Chronic Prednisone Treatment

Prednisone is often used for the treatment of autoimmune and inflammatory diseases but they suffer from variable therapeutic responses and significant adverse effects. Serum biological markers that are modulated by chronic corticosteroid use have not been identified. Myasthenia gravis is an autoimmune neuromuscular disorder caused by antibodies directed against proteins present at the post-synaptic surface of neuromuscular junction resulting in weakness. The patients with myasthenia gravis are primarily treated with prednisone. We analyzed the metabolomic profile of serum collected from patients prior to and after 12 weeks of prednisone treatment during a clinical trial. Our aim was to identify metabolites that may be treatment responsive and be evaluated in future studies as potential biomarkers of efficacy or adverse effects. Ultra-performance liquid chromatography coupled with electro-spray quadrupole time of flight mass spectrometry was used to obtain comparative metabolomic and lipidomic profile. Untargeted metabolic profiling of serum showed a clear distinction between pre- and post- treatment groups. Chronic prednisone treatment caused upregulation of membrane associated glycerophospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, 1, 2-diacyl-sn glycerol 3 phosphate and 1-Acyl-sn-glycero-3-phosphocholine. Arachidonic acid (AA) and AA derived pro-inflammatory eicosanoids such as 18-carboxy dinor leukotriene B4 and 15 hydroxyeicosatetraenoic acids were reduced. Perturbations in amino acid, carbohydrate, vitamin and lipid metabolism were observed. Chronic prednisone treatment caused increase in membrane associated glycerophospholipids, which may be associated with certain adverse effects. Decrease of AA and AA derived pro-inflammatory eicosanoids demonstrate that immunosuppression by corticosteroid is via suppression of pro-inflammatory pathways. The study identified metabolomic fingerprints that can now be validated as prednisone responsive biomarkers for the improvement in diagnostic accuracy and prediction of therapeutic outcome.     

9G4+ Antibodies Isolated from HIV-Infected Patients Neutralize HIV-1 and Have Distinct Autoreactivity Profiles

Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these “9G4+” antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.