Involvement of serum vascular endothelial growth factor family members in the development of obesity in mice and humans

Adipose tissue is highly vascularized implying that angiogenesis takes place in its expansion. The aim of this study was to compare the concentrations of members of the vascular endothelial growth factor (VEGF) family in obesity. Serum concentrations of VEGFs were analyzed in 15 lean (BMI 20.3±2.5 kg/m²) and 24 obese (BMI 47.6±5.9 kg/m²) volunteers. Obese patients showed significantly increased circulating VEGF-A (150±104 vs. 296±160 pg/ml; P<.05), VEGF-B (2788±1038 vs. 4609±2202 arbitrary units; P<.05) and VEGF-C (13 453±5750 vs. 17 635±5117 pg/ml; P<.05) concentrations. Interestingly, levels of VEGF-D were reduced in obese individuals (538±301 vs. 270±122 pg/ml; P<.01). In addition, VEGF-A significantly decreased after weight loss following Roux-en-Y gastric bypass (BMI from 46.0±8.0 to 28.9±4.2 kg/m² P<.0001 vs. initial) from 345±229 to 290±216 pg/ml (P<.01). Moreover, in order to corroborate the human findings VEGF-A levels were analyzed during the expansion of adipose tissue in two dynamic models of murine obesity. Serum VEGF-A was significantly increased after 12 weeks on a high-fat diet (43.3±9.0 vs. 29.7±9.1 pg/ml; P<.01) or in ob/ob mice (52.2±18.0 vs. 29.2±7.7 pg/ml; P<.01) and was normalized after leptin replacement in the latter (32.4±14.0 pg/ml; P<.01 vs. untreated ob/ob). Our data indicates the involvement of these factors in the expansion of adipose tissue that takes place in obesity in relation to the need for increased vascularization, suggesting that manipulation of the VEGF system may represent a potential target for the pharmacological treatment of obesity.     

Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova

In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l⁻¹ for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l⁻¹ and 2.8 ± 0.16 μg l⁻¹ for L and H ewes, respectively, during the 12 day priming period (P < 0.001). Eight hours after progesterone withdrawal, levels had fallen to 0.9 ± 0.06 μg l⁻¹ and 0.8 ± 0.07 μg l⁻¹, respectively, then rose to 17.8 ± 3.01 μg l⁻¹ and 12.9 ± 2.50 μg l⁻¹ (P > 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r² = 0.79, P < 0.001), the interval to oestrous onset (r² = 0.52, P < 0.01) and timing of the LH surge (r² = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.     

Differences in Clinical Manifestations and Tumor Features Between Metastatic Pheochromocytoma/Paraganglioma Patients With and Without Germline SDHB Mutation

ObjectiveTo compare metastatic pheochromocytoma/paraganglioma (MPP) patients with germline SDHB mutations (SDHB MPP) and without SDHB mutations (non-SDHB MPP) in terms of baseline clinical manifestations, tumor characteristics, and outcomes.MethodsClinical data were retrospectively reviewed in 101 MPP patients, including 34 SDHB MPP patients and 61 non-SDHB MPP patients.ResultsSDHB MPP patients presented at a younger age at onset, diagnosis, or metastasis (25 ± 16 vs 36 ± 14, 28 ± 17 vs 38 ± 15, and 31 ± 17 vs 44 ± 14 years old, respectively, P < .01 for all) than non-SDHB patients. Compared with their non-SDHB counterparts, SDHB patients were more likely to have paragangliomas (83% vs 47%, P < .05), synchronous metastases (44% vs 23%, P < .05), bone metastases (80% vs 48%, P < .01), and a shorter progression-free survival (3 years vs 5 years, P < .01). The Ki-67 index was higher in SDHB tumors (P < .05). The 5- and 10-year survival rates were 79% and 74%, respectively, in all patients. Seventeen patients died from MPP, and the time from metastasis to death in patients who had received systemic therapy was significantly longer than in those who had not (3.1 ± 1.5 vs 1.4 ± 0.7 years, P < .01).ConclusionCompared with MPP patients without SDHB mutations, MPP patients with SDHB mutations were younger at onset, diagnosis, or metastasis; had a higher incidence of synchronous metastases, higher ratio of paraganglioma, and higher Ki-67 index; had a shorter postoperative progression-free survival; and were more likely to develop bone metastasis or sole liver metastasis. Our results suggest that patients with SDHB mutations should be identified early and monitored regularly to achieve optimal clinical outcomes.     

Nordic Walking Improves Cardiometabolic Parameters,Fitness Performance,and Quality of Life in Older Adults With Type 2 Diabetes

ObjectiveTo assess the effect of Nordic walking (NW) on cardiometabolic health, physical performance, and well-being in sedentary older adults with type 2 diabetes (T2D).MethodsFifteen subjects with T2D (female, 5; male, 10; age, 65 ± 6.2 years [mean ± standard deviation]; body mass index, 27.3 ± 4.9 kg/m² [mean ± standard deviation]) were enrolled in a 6-month NW training program. The fasting glucose and glycosylated hemoglobin levels, lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides), systolic blood pressure (SBP), and diastolic blood pressures were measured before and after the intervention. Participants’ quality of life (Short-Form Health Survey) and physical fitness (6-minute walking test) were also evaluated.ResultsCompared with baseline, NW significantly improved the fasting glucose level (103.5 ± 18.5 vs 168.7 ± 37.7 mg/dL, P = .01), SBP (121.8 ± 12.2 vs 133 ± 14.4 mm Hg, P = .02), physical fitness (759.88 ± 69 vs 615.5 ± 62.6 m, P < .001), and both mental health (54.5 ± 4.4 vs 45.7 ± 5.6, P < .01) and physical health (49.8 ± 4.7 vs 40.3 ± 5.9, P < .01). The levels of glycosylated hemoglobin (6.15% ± 0.8% vs 6.4% ± 1%, P = .46), total cholesterol (162.2 ± 31.2 vs 175.5 ± 28.8 mg/dL, P = .13), low-density lipoprotein cholesterol (95.2 ± 24.2 vs 106.3 ± 32.3 mg/dL, P = .43), and triglycerides (135.5 ± 60.8 vs 127.6 ± 57.4 mg/dL, P = 0.26) improved without reaching significance.ConclusionNW training improved the glycemic levels, SBP, physical fitness, and perception of quality of life in older adults with T2D. NW represents a suitable complementary strategy to improve the global health status in this population.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

Production of hand-made cloned buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) embryos from non-viable somatic cells

Use of non-viable somatic cells for hand-made cloning (HMC) can enable production of cloned animals from tissues obtained from elite or endangered dead animals. Buffalo skin fibroblast cells were rendered non-viable by heat treatment and used for HMC. Although fusion (93.6 ± 1.72 vs 67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate was lower (P < 0.001) than that for controls, blastocysts could be successfully produced. However, blastocyst rate (34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs 10.9 ± 1.21) was higher (P < 0.001) than that of controls. In another experiment, ear tissue of slaughterhouse buffaloes was preserved in mustard oil at room temperature for 48 h following which somatic cells were harvested by enzymatic digestion and used for HMC. Although fusion (96.8 ± 1.48 vs 84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower (P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were similar to those of controls. In conclusion, HMC can be successfully used for production of blastocysts from non-viable cells and from cells obtained from freshly slaughtered buffaloes. This can pave the way for the restoration of farm or wild animals by HMC if somatic cells could be obtained within a few hours after their death.     

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8⁺ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Hemodialysis Decreases Serum Brain-Derived Neurotrophic Factor Concentration in Humans

In the present study we have evaluated the effect of a single hemodialysis session on the brain-derived neurotrophic factor levels in plasma [BDNF]_pl and in serum [BDNF]_s as well as on the plasma isoprostanes concentration [F₂ isoprostanes]_pl, plasma total antioxidant capacity (TAC) and plasma cortisol levels in chronic kidney disease patients. Twenty male patients (age 69.8?±?2.9?years (mean?±?SE)) with end-stage renal disease undergoing maintenance hemodialysis on regular dialysis treatment for 15?C71?months participated in this study. A single hemodialysis session, lasting 4.2?±?0.1?h, resulted in a decrease (P?=?0.014) in [BDNF]_s by ~42?% (2,574?±?322 vs. 1,492?±?327?pg?ml^?1). This was accompanied by an increase (P?<?10^?4) of [F₂-Isoprostanes]_pl (38?±?3 vs. 116?±?16?pg?ml^?1), decrease (P?<?10^?4) in TAC (1,483?±?41 vs. 983?±?35 trolox equivalents, ??mol?l^?1) and a decrease (P?=?0.004) in plasma cortisol level (449.5?±?101.2 vs. 315.3?±?196.3?nmol?l^?1). No changes (P?>?0.05) in [BDNF]_pl and the platelets count were observed after a single dialysis session. Furthermore, basal [BDNF]_s in the chronic kidney disease patients was significantly lower (P?=?0.03) when compared to the age-matched control group (n?=?23). We have concluded that the observed decrease in serum BDNF level after hemodialysis accompanied by elevated [F₂-Isoprostanes]_pl and decreased plasma TAC might be caused by enhanced oxidative stress induced by hemodialysis.     

The effects of chronic environmental stress on parturition and on oxytocin and vasopressin secretion in the pig

Previous work has suggested that an acute behavioural confinement in mid-partum can inhibit oxytocin secretion and prolong delivery in the pig, an effect that is opioid mediated. The present experiment investigated the effect of longer-term (chronic) behavioural confinement, that has previously been shown to elevate total plasma cortisol, on speed of delivery and on plasma oxytocin and lysine vasopressin concentrations during the peri-parturient period in primiparous pigs (gilts). Five days before their expected parturition (farrowing) date, gilts with preplaced jugular catheters were either confined to farrowing crates that severely restricted maternal behaviour, or housed in pens that permitted free movement and maternal behaviour (e.g. nest building). Blood samples were taken continuously from 24 h before the birth of the first piglet (BFP) to 6 h post-BFP, and for oxytocin on Days 1, 2, and 7 following parturition (Days P1, P2, P7). Both oxytocin and vasopressin were strongly influenced by parturition (P<0.001). There was no overall effect of chronic crating on either hormone, but crated and penned gilts did show significant differences with respect to the pattern of both oxytocin and vasopressin concentrations over time (P<0.05 in both cases). Oxytocin and vasopressin first increased in crated and penned gilts from 3 h pre-BFP (P<0.05). Crated gilts subsequently showed greater increases in both oxytocin and vasopressin over the first hour of delivery than penned gilts (mean oxytocin (pmol 1⁻¹): 53.3±8.5 vs. 39.7±5.0 for crated vs. penned gilts; mean vasopressin (pmol l⁻¹):4.4±0.7 vs. 2.0±04 for crated vs. penned gilts; both P<0.05). For oxytocin, crated gilts then showed subsequent declining concentrations relative to penned gilts (P<0.05). For vasopressin, penned gilts reached similar concentrations as crated gilts in the third hour post-BFP before vasopressin concentrations in both groups declined. Crated gilts also gave birth to piglets faster in the early stages of delivery (e.g. mean interval between Piglets 2 and 3 (min): 9.6±2.5 vs. 25.6±8.54 for crated and penned gilts, respectively: P<0.02). We conclude that confinement of gilts to a farrowing crate for 5 days neither adversely affects the progress of delivery in the primiparous pig nor the secretion of posterior pituitary hormones involved in parturition.     

Short-term inhibition of prolactin secretion by naloxone treatment in the pregnant gilt

The involvement of endogenous opioids in modulation of prolactin (PRL) secretion during pregnancy in the pig was studied. Twenty-four crossbred pregnant gilts (150 ± 10 kg) were cannulated via the cephalic vein 24–48 h before treatment with 1 mg kg⁻¹ body weight of naloxone (NAL) or 3 ml of saline (CONT) i.v. at Day 40 (NAL, n = 6; CONT, n = 6) or Day 70 (NAL, n = 6; CONT, n = 6) of pregnancy. Blood plasma was collected at 15 min intervals from 1 h before to 3 h after treatment with NAL or saline. At Day 40 of pregnancy, administration of NAL caused a decrease in mean plasma PRL concentrations at 60 min, 120 min and 180 min post-treatment (NAL, 19.1 ± 1.3 ng ml⁻¹, P < 0.05; 15.8 ± 0.6 ng ml⁻¹, P < 0.001; 14.6 ± 0.7 ng ml⁻¹, P < 0.001, respectively) when compared with the CONT group (22.9 ± 0.7 ng ml⁻¹, 21.6 ± 0.6 ng ml⁻¹ and 22.4 ± 0.5 ng ml⁻¹, respectively). Mean plasma estradiol concentration was higher (P < 0.01) in the NAL group during the second and third hour post-treatment than in the CONT group. At Day 70 of pregnancy, infusion of NAL also decreased (P < 0.001) plasma PRL concentrations at 60 min, 120 min and 180 min after treatment (20.1 ± 1.6 ng ml⁻¹, 16.2 ± 1.5 ng ml⁻¹ and 14.8 ± 0.4 ng ml⁻¹, respectively) compared with the CONT group (33.4 ± 1.7 ng ml⁻¹, 34.1 ± 1.3 ng ml⁻¹ and 29.1 ± 0.9 ng ml⁻¹, respectively). Estradiol concentrations were not different (P > 0.05) between groups in this stage of gestation. Mean concentrations of progesterone were similar during the pre- and post-treatment periods in both stages of pregnancy.These data would suggest a possible role of the opioids in modulation of PRL secretion at these stages of pregnancy in the pig.     

Clinical Profile of Nonalcoholic Fatty Liver Disease Among Young Patients With Type 1 Diabetes Mellitus Seen at a Diabetes Speciality Center in India

ObjectiveTo estimate the prevalence and clinical profile of nonalcoholic fatty liver disease (NAFLD) among young type 1 diabetes mellitus (T1DM) patients at a tertiary care diabetes center in India.MethodsElectronic medical records of T1DM patients (age at first diagnosis of T1DM ≤ 25 years) registered between January 1992 and May 2013 who had undergone ultrasonography and denied history of any alcohol intake (n = 736) were reviewed. NAFLD was diagnosed if there was any degree of fatty liver. Retinopathy was initially assessed by direct and indirect ophthalmoscopy and later by retinal photography. Nephropathy was diagnosed if urine protein excretion was > 500 mg/day, and neuropathy was diagnosed if a patient’s vibration perception threshold on biothesiometry was ≥ 20 V.ResultsA total of 204/736 (27.7%) T1DM patients had NAFLD. Compared to T1DM subjects without NAFLD those with NAFLD had higher body mass index (BMI) (18.9 ± 4.2 vs. 20.2 ± 4.7 kg/m², P < .001), waist circumference (67.9 ± 13.2 vs. 71.9 ± 13.3 cm, P < .05), systolic blood pressure (110 ± 15 vs. 116 ± 18 mm Hg, P < .001) and diastolic blood pressure (72 ± 9 vs. 74 ± 10 mm Hg, P < .05), while fasting blood glucose (201 ± 101 vs. 183 ± 101 mg/dL, P < .05) and alkaline phosphatase (419 [12.5] vs. 315 [15.8], P < .001) levels were lower in patients with T1DM with NAFLD. Multiple logistic regression analysis showed a significant association between NAFLD and retinopathy (odds ratio [OR]: 2.01, 95% confidence interval [CI]: 1.13-3.43; P = .017, after adjusting for sex, duration of diabetes, overweight/obesity, hypertension, fasting plasma glucose, nephropathy, and nephropathy (OR: 1.89, 95% CI: 1.02-3.50; P = .042), after adjusting for sex and fasting plasma glucose.ConclusionsThis study suggests that NAFLD is also seen among T1DM patients and that it has an independent and significant association with retinopathy and nephropathy. (Endocr Pract. 2014;20:1249-1257)     

Melanopsin-expressing retinal ganglion cell loss and behavioral analysis in the Thy1-CFP-DBA/2J mouse model of glaucoma

In this study,the role of melanopsin-expressing retinal ganglion cells(mRGCs) in the glaucoma-induced depressive behavioral response pattern was investigated.The CFP-D2 transgenic glaucoma animal model from five age groups was used in this study.Immunohistochemical labeling,quantitative analysis of mRGC morphology,open field test(OFT),and statistical analysis were used.In comparison with C57 BL/6 mice,the age-matched CFP-D2 mice had significantly elevated intraocular pressure(IOP).We observed parallel morphological changes in the retina,including a reduction in the density of cyan fluorescent protein(CFP) expressing cells(cells mm 2 at 2 months of age,1309±26;14 months,878±30,P<0.001),mRGCs(2 months,48±3;14 months,19±4,P<0.001),Brn3b-expressing RGCs(2 months,1283±80;14 months,950±31,P<0.001),Brn-3b expressing mRGCs(5 months,50.17%±5.5%;14 months,12.61%±3.8%,P<0.001),and reduction in the dendritic field size of mRGCs(mm2 at 2 months,0.077±0.015;14 months,0.065±0.015,P<0.05).CFP-D2 mice had hyperactive locomotor activity patterns based on OFT findings of the total distance traveled,number of entries into the center,and time spent in the center of the testing apparatus.The glaucoma induced hyperactive response pattern could be associated with dysfunctional mRGCs,most likely Brn-3b-positive mRGCs in CFP-D2 mice.     

Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase

Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (control) 0.5 mM cysteine prior to washing and suspension in fresh cysteine-free media. Cysteine incubated cells had higher intracellular cysteine levels compared to controls (9.6 ± 0.78 vs. 6.5 ± 0.65 nmol/mg protein, P < 0.02, n = 6 ± SE). Cell homeostasis indicators were similar in the two groups. Cysteine incubated cells had significantly higher glutathione peroxidase (GPx) activity (1.11 ± 0.23 vs. 0.54 ± 0.1 U/mg protein, P < 0.05, n = 5 ± SE) and significantly greater expression of GPx-1 (5.01 ± 0.48 vs. 3.01 ± 0.25 OD units/mm², P < 0.05, n = 4 ± SE) compared to controls. Upon exposure to H₂O₂, cysteine incubated cells generated fewer reactive oxygen species and took longer to show contractile changes and undergo hypercontracture. However, when cells were exposed to H₂O₂ in the presence of 0.05 mM of the GPx inhibitor mercaptosuccinic acid, this increased the control cells’ susceptibility to H₂O₂ and completely abolished the cysteine mediated protection. These results suggest a new role for cysteine in myocardial protection involving stimulation of glutathione peroxidase.     

Proinflammatory conditions promote hepatocellular carcinoma onset and progression via activation of Wnt and EGFR signaling pathways

The aim of the current study was to investigate how proinflammatory conditions affect growth and progression of hepatocellular carcinoma. Human hepatoma cell lines were treated with lipopolysaccharide (LPS) or cyclooxygenase-2 inhibitor, Celecoxib, and in vitro proliferation, apoptosis, and cell cycle progression were assessed. This was followed up with in vivo xenograft assays to monitor tumor growth and metastatic progression under different treatment conditions. While LPS induced cell proliferation, Celecoxib induced apoptosis. Flow cytometry analysis demonstrated that S-phase cell count in LPS group was higher than control group (41.9 ± 3.2 vs 30.6 ± 0.1 %, respectively), whereas G0/G1-phase cells were significantly higher in the Celecoxib group in comparison with the control group (69.6 ± 5.0 vs 50.4 ± 1.6 %, respectively) (p < 0.05). Immunoblot analyses showed induction of epidermal growth factor receptor expression and induction and nuclear accumulation of Wnt/β-catenin and p65 in LPS group. Xenograft assays showed that LPS treatment induced comparatively large, rapidly growing tumors (2,702 ± 572 mm³) that metastasized to lungs, whereas Celecoxib treatment alone (1,008 ± 296 mm³) or in combination with LPS (1,303 ± 283 mm³) suppressed tumor growth in comparison to control groups (2,072 ± 456 mm³) (n = 5; p < 0.05). Inflammation can thus promote hepatoma cell proliferation and growth, and enhance the invasion and metastatic ability of hepatocarcinoma cells through inducing tumor angiogenesis, which in turn may be related to the activation of Wnt/β-catenin and EGFR signaling pathways.     

The effects of Δ9-tetrahydrocannabinol Δ9-THC) on the regional concentrations of spermidine in the rat brain

The effects of intravenous administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC, 2 mg/kg, i.v.) on the regional brain spermidine concentrations of the rat were examined. Thirty minutes after vehicle treatment, the spermidine concentrations were: for the medulla oblongata/pons, 68.2 ± 7.7 μg/g; the hypothalamus, 67.7 ± 2.6 μg/g; the midbrain, 59.1 ± 4.4 μg/g; the cerebellum, 47.3 ± 5.9 μg/g and for the cortex, 13.8 ± 0.8 μg/g. Thirty minutes after Δ⁹-THC, these concentrations were reduced in the midbrain (47.0 ± 8.0% of control, P < 0.0001) and cortex (69.4 ± 7.4% of control, P < 0.009). The spermidine concentrations were not significantly altered in the medulla oblongata/pons (86.5 ± 13.3%, P > 0.36), hypothalamus (107.2 ± 11.8, P > 0.36) or cerebellum (89.0 ± 14.4%, P < 0.48). These results suggest that spermidine within the midbrain and cortex may be involved in the expression of some of the actions of Δ⁹-THC.     

Long-Term,Sustained, Lifestyle-Induced Weight Loss in Severe Obesity: the Get-Real Program

Objective: To study the long-term effectiveness of a patient-centered, multidisciplinary lifestyle intervention treatment in patients medically eligible for bariatric surgery.Methods: Using a case-control study design, we compared treatment results for 98 adults (mean body mass index [BMI], 44.2 kg/m²) with the outcomes of 148 controls (mean BMI, 43.0 kg/m²) receiving standard care. The approach included a phased triage for inclusion, followed by 12 lifestyle intervention group sessions alternating with individual visits for behavior, diet, and exercise instructions.Results: At 2 years, weight loss averaged 15.3 ± 1.4 kg (P<.0010) (12 ± 1% of initial body weight [IBW], P<.001; 21 ± 2% of excess body weight [EBW], P<.001) in an intention-to-treat (ITT) analysis; in completers, weight loss was 18.8 ± 1.5 kg (P<.001) (15 ± 1% IBW, P<.001; 26 ± 3% EBW, P<.001). A total of 42 patients lost ≥10% IBW. Controls remained weight stable (P =.35); 3% lost ≥10% IBW. Patients achieving weight loss that would be considered satisfactory for bariatric surgery included 20% who achieved ≥35% EBW loss, 29% who achieved a BMI <35 kg/m² (if starting BMI <50 kg/m²) or BMI <40 kg/m² (if starting BMI ≥50 kg/m²), and 37% who achieved EBW loss ≤50%. These values for completers were 31, 39, and 48%, respectively. In the 55 patients starting the program ≥4 years ago, weight loss maintenance of 12 ± 1% IBW (ITT, 16 ± 1% in completers) was observed.Conclusion: Substantial nonsurgical weight loss, maintained at 2 to 4 years, is achievable in severely obese patients using comprehensive lifestyle approaches; the efficacy/safety trade-off in obesity treatment is an important consideration in interpreting these results.Abbreviations: BMI = body mass index EBW = excess body weight HbA_1c = glycated hemoglobin IBW = initial body weight LOCFA = last observation carried forward analysis