OBJECTIVETo primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.

METHODSForty patients with severe burn admitted to our ward from March 2013 to October 2015, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and ulinastatin treatment group (UT, n=20) according to the random number table and patient's consent. After admission, patients in group CT received antishock treatment, antibiotic treatment, debridement, skin grafting, and nutrition support, etc. On the basis of the above-mentioned treatment, patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day, with a dosage of 8×10(5) U every time, for 7 days in addition. Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1, 3, 5 and 7, respectively. Twenty healthy volunteer were selected as health control group (HC), and peripheral venous blood samples were collected on the first day of the study. Percentage of CD4(+) CD25(+) regulatory T lymphocytes (Tregs) was determined by flow cytometer. The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value). Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes, and content of IL-4 and γ interferon (IFN-γ) in serum were detected by enzyme-linked immunosorbent assay. Expression of human leukocyte antigen-DR (HLA-DR) on CD14(+) monocytes was determined by flow cytometer. Data were processed with analysis of variance for repeated measurement, chi-square test, and LSD-t test.

RESULTS(1) Compared with that of volunteer in group HC, the percentage of CD4(+) CD25(+) Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043, P values below 0.01). Compared with that in group CT, the percentage of CD4(+) CD25(+) Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071, P values below 0.01). (2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1, 3, 5, and 7 was respectively 0.71±0.11, 0.61±0.15, 0.54±0.12, and 0.67±0.17, which was significantly lower than that in group HC (1.21±0.22, with t values from 8.686 to 11.957, P values below 0.01). The proliferative activity of T lymphocytes of patients in group UT on PTD 3, 5, and 7 were respectively 0.81±0.11, 0.85±0.14, and 1.08±0.13, which was significantly higher than that in group CT (with t values from 4.808 to 8.568, P values below 0.01). (3) Compared with those of volunteer in group HC, content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288, P values below 0.01), content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451, P values below 0.01), and content of IFN-γ in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659, P values below 0.01). Compared with those in group CT, content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869, P values below 0.01), content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843, P values below 0.01), and content of IFN-γ in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521, P values below 0.01). (4) The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group CT on PTD 1, 3, 5, and 7 were respectively (28±6)%, (25±7)%, (25±7)%, and (39±10)%, which were significantly lower than the percentage of volunteer in group HC [(87±8)%, with t values from 16.323 to 25.645, P values below 0.01]. The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group UT on PTD 3, 5, and 7 were respectively (40±6)%, (42±9)%, and (49±10)%, which were significantly higher than those in group CT (with t values from 3.071 to 7.324, P values below 0.01).

CONCLUSIONSOn the basis of CT, additional ulinastatin intervention can decrease CD4(+) CD25(+) Tregs percentage, improve the immune function of T lymphocytes and T helper cells, and increase expression of HLA-DR on CD14(+) monocytes of patients with severe burn injury, thus improve the immune function of patients.

Burns ; drug therapy ; immunology ; Cells, Cultured ; Debridement ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; therapeutic use ; Humans ; Interferon-gamma ; blood ; Interleukin-2 ; metabolism ; Interleukin-4 ; blood ; Monocytes ; immunology ; Skin Transplantation ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVE: To evaluate the immunogenicity of self-made sheep skin acellular dermal matrix (ADM).
 METHODS: Both cross-linked and non-crosslinked ADMs were made after the sheep skin ADM was ready. A total of 160 healthy mice were then randomly divided into 4 groups. Group A, cross-linked ADM was implanted under skin; Group B, non-crosslinked ADM was implanted under skin; Group C, allosome skin was embedded under skin; Control group, without any implantation. Any visible changes in implantation region and any apparent rejection was recorded. Any symptoms of inflammatory reaction in implantation region were also observed by microscope. The levels of IL-4 and IFN-γ in serum were measured on the 3rd day, 1 week, 2 weeks and 3 weeks after the operation, respectively.
 RESULTS: Visual study: wound healing in implantation regions were obvious, the immunologic rejection was not significant, and the sheep ADM was absorbed gradually by ambient tissues. Microscope results: the connection between the sheep ADM and surrounding tissues was very tight with infiltration of a few inflammatory cells into the depth of sheep ADM. The structure of sheep ADM was gradually destroyed by inflammatory cells. The levels of IL-4 and IFN-γ were not significant difference between the group of A, B or C and the control group on the 3rd day after the operation. The inflammatory reaction in implantation region of the control group degraded after 3 days, concomitant with a decrease in the levels of IL-4 and IFN-γ. But the levels of IL-4 and IFN-γ in the Group A, B or C remained at high level, suggesting the elevation of IL-4 and IFN-γ levels were related to the immunologic reaction by sheep ADM. The values of IL-4/IFN-γ were significant difference between the group A, B or C and the control group at the end of the 1st week, 2nd week, and 3rd week. However, there were not significantly different among the Group A, B and C.
 CONCLUSION Sheep skin ADMs do not cause apparent rejection from mice after the transplantation because of the low immunogenicity. The cross-linked and non-crosslinked ADMs do not obviously affect the levels of IL-4 and IFN-γ.
Acellular Dermis ; Animals ; Graft Rejection ; blood ; Heterografts ; immunology ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Mice ; Random Allocation ; Sheep ; Skin Transplantation ; Wound Healing

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.
Animals ; Antigen Presentation ; Antigen-Presenting Cells/immunology/metabolism ; CD8-Positive T-Lymphocytes/*immunology ; Epitopes, T-Lymphocyte/*immunology ; Female ; Graft Rejection/*immunology ; Interferon-gamma ; Lymphocyte Activation/immunology ; Lymphocyte Count ; Mice ; Minor Histocompatibility Antigens/*immunology/metabolism ; *Skin Transplantation ; Transplantation, Homologous

To explore the role of dentritic epidermal T lymphocytes ( DETCs) in immune rejection of skin allograft in mice and its related mechanism. Methods (1) Full-thickness skin was harvested from back of one male wild type (WT) C57BL/6 mouse. Epithelial cells were isolated for detection of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morphological characteristics of DETCs with immunofluorescence technology. (2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice, 7 female WT C57BL/6 mice (group WT), and 7 female ybT lymphocytes 8 gene knock-out (GK) C57BL/6 mice (group GK) were used. Full-thickness skin in the size of 1.4 cm x 1.4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm x 1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. (3) Two male WT C57BL/6 mice were used to isolate epithelial cells. Cells were inoculated into 48-well plate and divided into activation group (A) and control group (C) according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 pL concanavalin A in the concentration of 2 microg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expression of interferon y in DETCs was detected with flow cytometer. (4) Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon gamma neutralizing group (IN) and control group (C) according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part (2). Before surgery and 72 hours after, mice in group IN were intraperitoneally injected with 200 pL interferon y neutralizing antibody in the concentration of 1 mg/mL, and those in group C with normal saline in the same volume as that in group IN. The survival time of skin grafts was observed and recorded using the methods in part (2), and the result of group IN was compared with that of group GK in part (2). The survival curve of skin grafts was processed with Log-rank ( Mantel-Cox) test. Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27%, and they were all CD3 cells. DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology. (2) The survival time of skin grafts of mice in group GK was 22-35 d, obviously longer than that in group WT (12-16 d, y2 = 14. 10 , P < 0.001). (3) Expression of interferon gamma was detected in 22. 70% DETCs in group A, which was obviously higher than that in group C (0.51%). (4) The survival time of skin grafts of mice in group IN was 19-24 d, which was obviously longer than that in group C (12-16 d, chi 2 = 13.60, P < 0.001) but close to that in group GK as in part (2) (chi2 = 0.06, P = 0.810). Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secretinf interferon gamma.
Allografts ; Animals ; Epidermis ; Female ; Graft Survival ; immunology ; physiology ; Interferon-gamma ; immunology ; metabolism ; Lymphocytes ; Male ; Mice ; Mice, Inbred C57BL ; Skin ; Skin Transplantation ; T-Lymphocytes ; immunology

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of gamma-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% --> 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.
Animals ; Bone Marrow Cells/cytology ; *Bone Marrow Transplantation ; Cell Proliferation ; Chimerism ; Cytokines/metabolism ; Gamma Rays ; Graft Survival ; *Immune Tolerance ; Killer Cells, Natural/immunology/radiation effects ; Leukocytes/immunology/radiation effects ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Animal ; Skin Transplantation ; T-Lymphocytes, Regulatory/cytology/*immunology/metabolism ; Whole-Body Irradiation

This study was aimed to evaluate whether human mesenchymal stem cell (MSC) and the Stro-1 positive subgroup have inducing immune tolerance effect in mouse skin graft model. Human MSC were isolated and cultured from bone marrow-derived mononuclear cells of healthy adults, and Stro-1 positive cells were sorted out. Female C57BL/6 mice and female BALB/c mice were respectively used as donors and recipients in skin allogenic graft model. The recipients were divided randomly into 4 groups: (1) Stro-1(+) MSC group: 2×10(6) Stro-1(+) MSC were injected into the irradiated recipient mice before skin graft. (2) MSC group: 2×10(6) MSC were injected into the irradiated recipient mice before skin graft. (3) Irradiation control group: the recipient mice were just irradiated before skin allogenic graft. (4) Congenic control group: the irradiated BALB/c mice received the skin from the congenic mice. The survival time and pathologic changes of skin grafts were observed by macro- and microscopy with HE staining. The transforming growth factor β1 (TGF-β1) concentration in plasma of recipient mice was measured by ELISA before and after grafting. The results indicated that the survival time of skin grafts in the MSC group was (12.13 ± 3.34) d, which was not notably longer than the irradiation control group (11.38 ± 1.01) d. The survival time of skin grafts was significantly prolonged in the Stro-1(+) MSC group (30.68 ± 5.89) d, as compared with the irradiation control group and the MSC group, respectively; the pathologic examination of skin grafts showed a clear structure. After grafting, the TGF-β1 concentration in plasma of recipient mice was almost the same as before grafting in the irradiation control group and the congenic control group, but it significantly increased in the MSC group and the Stro-1(+) MSC group. It is concluded that the Stro-1(+) MSC induce greater immune tolerance than the unsorted MSC, and significantly prolong the survival time of skin grafts in vivo, while TGF-β1 does not contribute to the immune tolerance.
Animals ; Female ; Graft Survival ; immunology ; Humans ; Immune Tolerance ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; methods ; Transforming Growth Factor beta1 ; blood ; Transplantation, Homologous

UNLABELLEDTo provide a reliable animal model for study of human CMV disease in gastrointestinal track, we tried to infect with murine cytomegalovirus (MCMV) in mice that were received allogenetic skin transplantation under immunosuppression. (1) Skin transplantation was performed between 18 donor C57BL/6 mice and 72 recipient BALB/c mice. (2) All recipient mice were then given Cyclosporine at 12 mg/kg daily for 2 weeks by intraperitoneal injection. Mice were randomly divided into 3 groups. Two experimental groups were received MCMV-infected mouse embryonic fibroblasts (MEF) at 10(4) PFU and 10(5) PFU respectively, and the control group received MEF only. We observed any possibly pathophysiological behavior changes and recorded the changes in body weight. The mice were sacrificed at 5d, 9d, 14d, 21d post infection and colon tissue was collected for analysis.

RESULTSMice infected with MCMV at 10(5) PFU group showed anorexia, lethargy and degression in locomotor activity. This group of mice showed significant decrease in body weight than that of other groups. Colon tissues were collected 14 days after infection. Histological examination revealed that the mucous layer became thinner in the proximal colon and increased number of lymphoid follicles in distal colon in infected animals. The changes in the mucosal structure was most prominent in the group 10(5) PFU MCMV. Viral DNA was present in the colon by in situ hybridization for IE1 gene, and viral gB transcript was positive by RT-PCR. One of the viral major proteins, pp65, was widely distributed in the colon by immunohistochemistry. These data demonstrated that MCMV established infection in colon of the mice after allogenetic skin transplantation. Electron microscopy showed that there were herpes virus particles in the colon tissue.

CONCLUSIONInfection with MCMV in mouse after allogenetic skin transplantation by nasal cavity inoculation resulted in the pathological changes in colon tissue similar to that of inflammation in human colon. The small animal model of colon inflammation may provide a platform for further study of pathogenesis as well as medical intervention of HCMV involved inflammation of human bowel.

Animals ; Colon ; immunology ; pathology ; virology ; Cytomegalovirus Infections ; immunology ; pathology ; virology ; Disease Models, Animal ; Female ; Herpesviridae Infections ; immunology ; pathology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Muromegalovirus ; genetics ; immunology ; isolation & purification ; Random Allocation ; Skin Transplantation ; adverse effects ; immunology ; pathology ; Transplantation, Homologous ; adverse effects ; immunology ; pathology ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo study the immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) in the allogeneic transplantation.

METHODSForty female C57BL/6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group, with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide (200 mg/kg, 2 d, q. d.) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 10(6) BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+, CD25+, Foxp3 and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA, including TGF-beta, IL-10 and IFN-gamma. T cells were co-cultured with 60Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay.

RESULTSThe skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29 (99.7%), CD44+ (96.7%), CD34- (1.6%), CD45- (1.3%). Compared with the control group and CP group, the ratio of the CD4+, CD25+, Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P < 0.05). Analysis of peripheral blood by ELISA showed significant high level of TGF-beta, IL-10 and low level of IFN-gamma in BMSCs group and CP group,compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased apparently in SD-BMSCs group and C57-BMSCs group (P < 0.05), but there was no significant difference between SD-BMSCs group and C57-BMSCs group (P > 0.05).

CONCLUSIONSThe immunotolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in the allogeneic transplantation might be associated with its effect on the proliferation of Treg cells and increasing expression of TGF-beta and IL-10, decreasing expression of IFN-gamma.

Animals ; Bone Marrow Cells ; immunology ; Female ; Immune Tolerance ; Interferon-gamma ; immunology ; Interleukin-10 ; immunology ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Rats ; Rats, Sprague-Dawley ; Skin Transplantation ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; immunology ; Transplantation, Homologous

Heat shock protein gp96 isolated from tumor tissues holds great promise for tumor immunotherapy. However, at present only very limited amount of gp96 protein can be isolated from tumor tissues. Here, we reconstituted the yeast-expressed gp96 (recombinant gp96, rgp96) with B16.F10 melanoma antigens in vitro to prepare new gp96 tumor vaccine on large-scale, and analyzed its induction of specific anti-tumor immunoresponses by ELISPOT, IFN-gamma intracellular staining and cytotoxicity assays. Immunization with rgp96-tumor antigen complexes significantly inhibited B16 tumor growth compared with either rgp96 or tumor antigens alone and led to enhancement of tumor-specific T-cell activities, which was found similar to that of tumor tissue derived gp96. Our results therefore may provide bases for large-scale preparation of the new generation of gp96 tumor vaccines.
Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; therapeutic use ; Female ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Melanoma, Experimental ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Skin Neoplasms ; therapy ; Yeasts ; genetics ; metabolism

BACKGROUNDEstrogen as well as CD4(+)Foxp3(+) regulatory T cells were shown to have a protective role not only in maintaining maternal-fetal tolerance but also against autoimmune diseases. We aimed to investigate whether the pregnancy levels of estrogen are enough to induce transplant tolerance as to maintain fetal-maternal tolerance.

METHODSWe established H-Y skin graft transplantation in C57BL/6 ovariectomized mice that reconstituted with estrogen. Subsequently, consecutive daily estrogen injection was administrated. Tregs and the cytokines in the peripheral blood were detected by flow cytometry and ELISA pre- and post-transplant.

RESULTSThe results indicated that pregnancy levels of estrogen could promote Tregs in secondary lymphoid organs and peripheral blood (P < 0.05) but not thymus (P > 0.05). The estrogen-treated recipients accepted H-Y skin grafts for more than 35 days (median survival time (MST): (44.0 ± 1.2) days) compared with estrogen-untreated mice (MST: (23.0 ± 1.6) days) (P < 0.05). It was also observed that estrogen up-regulated the expression of Foxp3, but did not affect CD3(+)CD8(+) effector T-cells in non-transplant mice. While in the presence of H-Y antigens, the expression of Foxp3 was more significant and CD3(+)CD8(+) effector T cells were decreased significantly (P < 0.05). Meanwhile, the up-regulated IL-10 and IL-4, and down-regulated IFN-γ could be observed (P < 0.05).

CONCLUSIONSPregnancy levels of estrogen may promote the conversion of peripheral Tregs in secondary lymphoid organs, but show no effect on the natural Tregs production, differentiation and maturity in central lymphoid organs. Furthermore, pregnancy levels of estrogen could significantly prolong the survivals of H-Y skin grafts by the expansion of Tregs, suppression of CD3(+)CD8(+) effector T-cells and immune shift towards Th2 cytokines.

Animals ; Cytokines ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Graft Survival ; drug effects ; H-Y Antigen ; immunology ; metabolism ; Immunohistochemistry ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-4 ; metabolism ; Mice ; Mice, Inbred C57BL ; Ovariectomy ; Pregnancy ; Skin Transplantation ; immunology ; T-Lymphocyte Subsets ; immunology