Control of IBMIR in Neonatal Porcine Islet Xenotransplantation in Baboons

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification

Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway

High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.Jared M. Campbell, Mark B. Nottle, Ivan Vassiliev, Megan Mitchell, and Michelle Lan

Modulation in voluntary neural drive in relation to muscle soreness

The aim of this study was to investigate whether (1) spinal modulation would change after non-exhausting eccentric exercise of the plantar flexor muscles that produced muscle soreness and (2) central modulation of the motor command would be linked to the development of muscle soreness. Ten healthy subjects volunteered to perform a single bout of backward downhill walking exercise (duration 30 min, velocity 1 ms−1, negative grade −25%, load 12% of body weight). Neuromuscular test sessions [H-reflex, M-wave, maximal voluntary torque (MVT)] were performed before, immediately after, as well as 1–3 days after the exercise bout. Immediately after exercise there was a −15% decrease in MVT of the plantar flexors partly attributable to an alteration in contractile properties (−23% in electrically evoked mechanical twitch). However, MVT failed to recover before the third day whereas the contractile properties had significantly recovered within the first day. This delayed recovery of MVT was likely related to a decrement in voluntary muscle drive. The decrease in voluntary activation occurred in the absence of any variation in spinal modulation estimated from the H-reflex. Our findings suggest the development of a supraspinal modulation perhaps linked to the presence of muscle soreness

Sow litter size is increased in the subsequent parity when lactating sows are fed diets containing n-3 fatty acids from fish oil

Supplementing diets with n-3 fatty acids from fish oil has been shown to improve reproductive performance in dairy cattle and sheep, but there is little published literature on its effects in sows. The aim of this study was to evaluate the reproductive performance of sows fed fish oil as a source of n-3 PUFA prefarrowing and during lactation. From d 107.7 ± 0.1 of pregnancy, 328 sows ranging in parity from 0 to 7 (parity 1.95 ± 0.09, mean ± SE) were fed either a diet containing tallow (control) or an isocaloric diet containing 3 g of fish oil/kg of diet (n-3). Diets were formulated to contain the same amount of DE (13.9 MJ/kg), crude fat (54 g/kg), and CP (174 g/kg). Sows were fed their treatment diet at 3 kg daily for 8 d before farrowing and continued on treatment diets ad libitum until weaning at 18.7 ± 0.1 d of lactation. After weaning, all sows were fed a gestation diet without fish oil until their subsequent farrowing. There was no effect (P > 0.310) of feeding n-3 diets prefarrowing on piglet birth weight, preweaning growth rate, piglet weaning weight, or sow feed intake. However, n-3 sows had a larger subsequent litter size (10.7 ± 0.3 vs. 9.7 ± 0.3 total born; 10.2 ± 0.3 vs. 9.3 ± 0.3 born live; P < 0.05). In conclusion, this is the first study to demonstrate that feeding sows a diet containing n-3 PUFA from fish oil fed before farrowing and during lactation increased litter size in the subsequent parity independent of energy intake.R. J. Smits, B. G. Luxford, M. Mitchell and M. B. Nottl

Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage

Reproductive responses to daily injections with porcine somatotropin before mating in gilts

Litter size and progeny birth weights are lower in gilts than in sows. Somatotropin (ST) is an important regulator of ovulation, fetal growth and survival. We therefore investigated effects of pST treatment of gilts for two to four weeks before mating on ovulation rate, behavioural estrus, fetal growth and survival, litter size and birth weights. In Experiment One, gilts were injected with 0, 30, 60 or 90 µg pST/kg/day for 14 days commencing 7 days after first estrus. Reproductive tracts were collected and corpora lutea and follicle numbers counted 5.5 days after second estrus. Ovulation rate (P=0.031) and number of medium-sized follicles (P=0.059) correlated positively with pST dose. In Experiment Two, gilts were injected with 0, 12.5, 25 or 50 µg pST/kg/day for 21 days from first estrus, and mated at second estrus. Numbers of corpora lutea, follicles and fetuses were counted at day 31 of pregnancy. Numbers of medium follicles and ovary weights were positively related to pST dose. In Experiment Three, 31 week old (1(st) replicate) or 27 week old (2(nd) replicate) gilts were injected daily with 0 or 12.5 µg pST/kg/day until mating 25.9 ± 0.6 days later, and delivered at term. Pre-mating pST increased total litter size in younger gilts in the 2(nd) replicate only (P<0.05). In conclusion, injecting gilts with pST before mating does not consistently alter ovulation rate, increases the number of medium follicles available for recruitment at the second mating after treatment and increases subsequent litter size in younger gilts.Kathryn L. Gatford, Christopher G. Grupen, Roger G. Campbell, Brian J. Luxford, Robert J. Smits, Phillip C. Owens and Mark B. Nottl