Proteasome Expression in Ovarian Heterotopic Allografts of Wistar and August Rats under Induction of Donor-Specific Tolerance

Russian Journal of Developmental Biology - The aim of this work is to investigate the dynamics for the ovarian tissue engraftment of inbred August rats transplanted to outbred Wistar rats and vice...     

Proteasomes on thyroid tissue allotransplantation under induction of donor-specific tolerance in rats

Proteasomes in the liver of August rats (RT1^c) were investigated 30 days after allotransplantation of Wistar rat (RT1^u) thyroid tissue under renal capsule with/without induction of donor-specific tolerance by donor splenocyte intraportal administration. The levels of total proteasome pool, immune proteasomes containing subunits LMP2 and/or LMP7, and proteasome regulators 19S and 11S were defined. Intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups, which indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of nontolerant rats compared to tolerant animals. In the liver of tolerant rats with accepted grafts, the number of mononuclear cells expressing the immune subunit LMP2 greatly increased in comparison with control and nontolerant animals. Study of accepted grafts showed an increase in the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them, compared to the tissue replacing the rejected grafts. Immune proteasomes were almost completely absent from the control intact thyroid tissue, while 19S/11S ratio was maximal in it. Thus, the development of the immune reaction or its suppression are accompanied by a change in the balance between different proteasome forms. Immune subunit LMP7 and 11S regulator are associated with the response against donor tissue. On the contrary, immune subunit LMP2 and 19S regulator are likely to be important for the development of immune tolerance and surviving tissue functioning. Immunofluorescence assay revealed a low content of the immune proteasomes in the follicle cells. Probably, formation of antigens for the major histocompatibility complex class I molecules was impaired by the low content of immune proteasomes, which led to immunological tolerance of hormone-producing follicle cells.     

Primary cell culture from pig neonatal thyroid gland: Growth, folliculogenesis, and hormone activity

A comprehensive assay of proliferative and hormonal activity in primary cell cultures derived from neonatal pig thyroid was carried out for the first time. Morphology and basal and thyroid-stimulating hormone (TSH)-stimulated secretion of thyroxin were evaluated in cultures, depending on the initial material placed into the culture: single cells or follicular conglomerates. Spontaneous and under chronic TSH stimulation folliculogenesis and formation of dome structures were assessed in culture. It was demonstrated that the cells expressed β-III-tubulin during prolonged cultivation with nerve growth factor.     

Changes in the proteasome function after induction of donor-specific tolerance in rats with ovarian allograft

Induction of donor-specific tolerance in a recipient is one of the methods for enhancing acceptance of the grafts of endocrine glands in the absence of immunodepressants, which interfere with hormone production. This paper describes changes in the proteasome pool in the rat liver, spleen, and graft during the development of donor-specific tolerance after intraportally infusing the recipient with donor splenocytes with subsequent allografting of ovarian tissue into the renal capsule. It has been demonstrated that the shift in the balance in the liver and graft proteasome pools towards the variants with the LMP2 subunit determines the development of immunological tolerance and graft retention. On the contrary, an increase in the forms with the LMP7 subunit induces the immune response and graft rejection.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.     

The development of the cell cryopreservation protocol with controlled rate thawing

Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing–thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath.     

Development of a cryopreservation protocol for testicular interstitial cells with the account of temperature intervals for controlled cooling below -60°С

A long course of anticancer therapy may lead to testicular steroidogenesis destruction. Cryopreservation of testicular interstitial cells (TIC) would be a strategy to protect hormonal and fertile potential of pre-pubertal boys treated with chemo – or radiotherapy. The aim of this research was to optimize protocols for freezing of TIC. Essential physical processes associated with the presence of dimethyl sulphoxide (Me₂SO) in the cryoprotectant solution take place at the temperatures below −60 °С. These processes are the eutectic crystallization at the stage of freezing and the recrystallization before the melting of the eutectic mixture at the stage of heating. Both of the processes affect the viability of the cells subjected to cryopreservation. Temperature intervals when these processes take place were determined by the method of thermoplastic deformation for 10% Me₂SO selected for cryopreservation of TIC. Rat TIC were cryopreserved using five different protocols which varied in cooling rates within the chosen temperature intervals. Post-thaw cell viability and metabolic activity were evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assays. Leydig cell recovery after cryopreservation was measured by 3-beta-hydroxysteroid dehydrogenase reaction. Based on the obtained results, the authors developed a cryopreservation protocol for TIC which makes it possible to achieve great cell viability due to using controlled cooling rates within the temperature intervals below −60 °С.     

Outcome of adrenal tissue fragments allotransplantation: The impact of cryopreservation

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4⁺ and CD8⁺ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.