Seasonal effects of central leptin infusion and prolactin treatment on pituitary SOCS-3 gene expression in ewes.

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 µg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

Seasonal effects of central leptin infusion on secretion of melatonin and prolactin and on SOCS-3 gene expression in ewes.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer-Locke buffer; 2) L1, roleptin, 0.5 microg/kg BW; and 3) L2, roleptin, 1.0 microg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.

Design and characterization of a tissue-equivalent CVD-diamond detector for clinical dosimetry in high-energy photon beams.

New solid-state detectors, based on chemical vapour deposited (CVD) polycrystalline diamonds produced by hot-filament (HF) or microwave plasma (MW) assisted deposition methods, were constructed for radiation therapy dosimetry. Properties of diamond crystals, such as high radiation sensitivity, resistance to radiation damage and tissue-equivalence giving a low-energy dependence are very advantageous for clinical dosimetry. Therefore the encapsulation was specially designed for these detectors to have as little influence as possible on the radiation response. The prototypes were irradiated with use of a wide range of photon beam qualities ((60)Co gamma-rays, 6 and 18 MV X-rays). The radiation sensitivity varied considerably between samples deposited with HF (9 nC Gy(-1)mm(-3)) and MW (66 and 144 nC Gy(-1)mm(-3)) methods. For all detectors the leakage current was of the order of 10% of the radiation-induced current (bias voltage 100 V, dose rate 0.3 Gy/min). When irradiated with (60)Co gamma-rays, the detectors showed a dose-rate linearity with an exponential Delta parameter close to unity. However, a difference of 8% was found between Delta values for the different beam qualities. A small energy dependence was observed, for which the most probable sources are interface effects due to the silver electrodes and partly the geometry of the encapsulation which needs to be further optimized. Despite some limitations in the performance of present prototype detectors, with an improved CVD technique producing crystals of better electrical and dosimetric properties, and with a well-designed tissue-equivalent encapsulation, CVD-diamonds could serve as very good dosimeters for radiotherapy.

Leptin as a nutritional signal regulating appetite and reproductive processes in seasonally-breeding ruminants.

Photoperiod and nutrition both exert major influences on reproduction. Thus, it seems axiomatic that seasonal rhythms in ovulation are influenced by nutrition. In this context, leptin is one of the most important hormonal signals involved in the control of energy homeostasis, feeding behavior and reproductive function in mammals. However, the number of published investigations establishing a functional interaction between leptin and photoperiodism in seasonal breeders is limited. In common with most seasonally-breeding mammals, sheep exhibit robust circannual cycles in body weight and reproduction, which are driven mainly by changes in day-length. Recently, attention has focused on the role of leptin in this process, particularly in its roles as a major peripheral signal controlling appetite, melatonin and prolactin secretion. The purpose herein is to review current concepts in the overall biology of leptin, to summarize its influence on the hypothalamic-pituitary axis, and to highlight recent developments in our understanding of its interaction with season in regulating appetite, body weight and reproduction in seasonally-breeding mammals. The latter observations may be important in delineating states of leptin resistance and obesity in humans.

NrCAM, a neuronal system cell-adhesion molecule, is induced in papillary thyroid carcinomas.

NrCAM (neuron-glia-related cell-adhesion molecule) is primarily, although not solely, expressed in the nervous system. In the present study, NrCAM expression was analysed in a series (46) of papillary thyroid carcinomas (PTCs) and paired normal tissues (NT). Quantitative reverse transcriptase (QRT)-PCR revealed that NrCAM expression was upregulated in all PTCs compared to normal thyroid, whatever the stage or size of the primary tumour. NrCAM transcript levels were 1.3- to 30.7-fold higher in PTCs than in NT. Immunohistochemistry (IHC) confirmed that the expression of NrCAM was considerably higher in tumours (score 2+/3+) than in adjacent normal paratumoural thyroid tissue. The NrCAM protein was detected in all but three (93.3%) PTC samples, and it was mainly cytoplasmic; in some cases there was additional membranous localisation - basolateral and partly apical. In the normal thyroid and tissues surrounding tumours, focal NrCAM immunolabelling was seen only in follicles containing tall cells, where staining was restricted to the apical pole of thyrocytes. Western blot analysis corroborated the QRT-PCR and IHC results, showing higher NrCAM protein levels in PTCs than in paired NT. The level of overexpression of the NrCAM mRNA in tumourous tissue appeared to be independent of the primary tumour stage (pT) or the size of the PTC. These data provide the first evidence that NrCAM is overexpressed in human PTCs at the mRNA and protein levels, whatever the tumour stage. Thus, the induction and upregulation of NrCAM expression could be implicated in the pathogenesis and behaviour of papillary thyroid cancers.

Influence of electrodes on the photon energy deposition in CVD-diamond dosimeters studied with the Monte Carlo code PENELOPE.

A new dosimeter, based on chemical vapour deposited (CVD) diamond as the active detector material, is being developed for dosimetry in radiotherapeutic beams. CVD-diamond is a very interesting material, since its atomic composition is close to that of human tissue and in principle it can be designed to introduce negligible perturbations to the radiation field and the dose distribution in the phantom due to its small size. However, non-tissue-equivalent structural components, such as electrodes, wires and encapsulation, need to be carefully selected as they may induce severe fluence perturbation and angular dependence, resulting in erroneous dose readings. By introducing metallic electrodes on the diamond crystals, interface phenomena between high- and low-atomic-number materials are created. Depending on the direction of the radiation field, an increased or decreased detector signal may be obtained. The small dimensions of the CVD-diamond layer and electrodes (around 100 microm and smaller) imply a higher sensitivity to the lack of charged-particle equilibrium and may cause severe interface phenomena. In the present study, we investigate the variation of energy deposition in the diamond detector for different photon-beam qualities, electrode materials and geometric configurations using the Monte Carlo code PENELOPE. The prototype detector was produced from a 50 microm thick CVD-diamond layer with 0.2 microm thick silver electrodes on both sides. The mean absorbed dose to the detector's active volume was modified in the presence of the electrodes by 1.7%, 2.1%, 1.5%, 0.6% and 0.9% for 1.25 MeV monoenergetic photons, a complete (i.e. shielded) (60)Co photon source spectrum and 6, 18 and 50 MV bremsstrahlung spectra, respectively. The shift in mean absorbed dose increases with increasing atomic number and thickness of the electrodes, and diminishes with increasing thickness of the diamond layer. From a dosimetric point of view, graphite would be an almost perfect electrode material. This study shows that, for the considered therapeutic beam qualities, the perturbation of the detector signal due to charge-collecting graphite electrodes of thicknesses between 0.1 and 700 microm is negligible within the calculation uncertainty of 0.2%.