Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Characterization of human CD55 and CD59 transgenic pigs and kidney xenotransplantation in the pig-to-baboon combination.

New transgenic pigs expressing combinations of regulators of complement activation and other molecules are needed to resist xenograft hyperacute rejection (HAR) and to further analyze and treat xenograft rejection. Double transgenic pigs for human CD55 (hCD55) and human CD59 (hCD59) using the promoter of the human elongation factor 1 alpha gene were generated, and their kidneys were transplanted into nonimmunosuppressed baboons. hCD55 and hCD59 were mainly expressed by the endothelial cells, and these cells showed increased resistance to complement-mediated lysis. Baboons receiving kidneys from hCD55hCD59 pigs survived for 5 and 6 days, and displayed alterations in coagulation. Thrombocytopenia and platelet microthrombi were present within the kidneys. Nontransgenic kidneys showed HAR in less than 2 days. Kidneys from pigs expressing hCD55hCD59 displayed protection against HAR in the absence of immunosuppression. Rejection was associated with coagulopathy leukocyte infiltration and a rebound of anti-alpha Gal antibodies.

Determination by echography of uterine changes around puberty in gilts and evaluation of a diagnosis of puberty.

Three experiments were carried out to evaluate the use of ultrasonography in assessing the onset of puberty in gilts. In experiment 1, gilts (n = 17) were scanned 3 times per week beginning at 133 and continuing until 187 days of age. The ultrasonic appearance of the uterus was described, quantified and compared with the reproductive status observed at slaughter. The quantification of the pictures showed a different correlation in time for infantile, impubertal, prepubertal and pubertal stages. For pubertal females, "uterine area" increased at around 180 days of age, well-defined sections of the uterine horns appeared 3 +/- 0.5 days before puberty. In infantile and impubertal gilts during the same period of age, uterine images remained dark and homogeneous; no significant change in the "uterine area" was observed. This difference in images allowed an evaluation of the diagnosis of puberty. In experiment 2, the gilts (n = 123) were scanned, the result was verified at slaughter the day after by examination of the genital tract. The uterine weight of the gilts that had reached a prepubertal or pubertal stage was significantly greater (P = 0.0001) than that in impubertal gilts. The sensitivity and the specificity of the diagnosis were 91.9% and 96.5% respectively. Experiment 3 was performed on a farm and echographic examinations were carried out one and five days after gilts (n = 117) arrived at the piggery. Oestrus detection or blood sampling for progesterone determinations were used as tools to determine the reproductive status. The sensitivity and the specificity of the diagnosis were 98.9% and 100% respectively. This diagnosis of puberty is thus accurate.