Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain

Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4⁺ pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4⁺ cells.     

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autoimmune encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4+ T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4+, CD8+, CD14+ (monocytes) and CD56+ NK cells but not to CD19+ B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.     

The influence of systemic growth hormone administration on the healing time of skin graft donor sites in a pig model

A more rapid healing of skin graft donor sites has often been observed during ultimoratio therapies with growth hormone in adults who have suffered extremely severe burns. The purpose of this animal experimental study was to examine the influence of systemic growth hormone administration on the healing time of skin graft donor sites under standardized conditions in pigs. The animals were 14 (7 experimental and 7 control) male, sexually mature, German domestic pigs, in which 30 skin graft donor sites 8 cm x 4 cm and 0.6 mm deep were created. Fifteen each of the skin graft donor sites were bandaged with the same material [hydrocolloid bandage (Varihaesive E) and PVP-iodine gauze (Braunovidon Gaze)]. The test period was 15 days for each pig, whereby recombinant growth hormone (0.5 IU/kg body weight per day) was applied subcutaneously in the experimental group. The bandages were changed under brief narcosis every 2 days, during which one skin-punch biopsy was taken per skin graft donor site, and blood samples were drawn for determination of the serum IGF-1 values. Photographic documentation was also recorded. The biopsies were examined histologically (hematoxylin and eosin stain) and immunohistochemically (collagen IV and VII, and laminin), whereby histologically the start of keratinization was assessed as a healing criterion. The serum IGF-1 values in the growth hormone group were statistically significantly higher than in the control group. Immunohistochemically, a complete basal membrane was observed in both the experimental and the control group after the 7th or 8th day. A clearly elevated serum IGF-1 level correlated in the growth hormone group with the skin graft donor sites healing. It could thus be demonstrated both clinically and histologically that systemic application of growth hormone results in a statistically significantly more rapid healing of the skin graft donor sites by 2 days earlier than in the control group.     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

Relationship between early pregnancy factor, mouse embryo-conditioned medium and platelet-activating factor.

The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.     

Enhanced survival of full-thickness skin grafts following the application of DC electrical fields

The present study was undertaken to determine if uses of exogenous electrical fields could improve the posttraumatic quality of dermis and epidermis in isolated grafts in the rat. In blinded procedures, a full-thickness area of skin was removed and reattached to the original site. A galvanic device delivering 4.5 microA of direct current was applied using three electrode orientations: (1) anode above graft (AT; N = 8), (2) cathode above graft (CT; N = 7), and (3) no current (NC; N = 7). Current delivery was discontinued 4 days postoperatively. Quantitative assessment at 7 days postoperatively indicated the presence of necrotic skin over 80 to 90 percent of the graft surface area in NC and CT animals. In contrast, only 50 percent of the graft area was necrotic in AT rats. Histologic examinations indicated a significantly thickened dermis in the AT (versus NC and CT) rats, accompanied by patches of multilayered intact epidermis, which was virtually absent from the other experimental groups. Results are consistent with the hypothesis that direct-current stimulation can affect skin graft survival and repair.     

Trypanosoma lewisi: Enhanced resistance in naive lactating rats and their suckling pups

Lactating rats and their suckling offspring were shown to have a naturally occurring resistance to Trypansoma lewisi which was greater than that seen in normal adult rats and was not dependent on previous exposure to the parasite. In the case of the pups maximum protection was dependent on suckling being the sole source of nutriment and also on the peak of the parasitemia falling within the nursing period. Supplementary solid food reduced survival rates in concurrent Hemobartonella muris—T. lewisi infections: pups infected at 10 days of age and totally dependent on nursing showed 82.5% survival but only 6.6% survival when solid food was allowed to supplement milk. However, supplementary food did not reduce survival in pups infected with only T. lewisi. When pups were totally dependent upon nursing until the normal time of weaning (21 days of age), infection with H. muris—T. lewisi at the following days of age allowed the indicated mean survival rates: 10 days (82.5%), 20 (32.5%), 30 (14.3%), and 40 (100%). Infection with T. lewisi alone at the following days of age allowed the indicated mean survival rates: 10 days (100%), 15 (76%), 17 (52%), 20 (100%), and 30 (100%). Lactating rat serum agglutinated “adult forms” of T. lewisi, which correlated well with the observed sudden resolution of parasitemia in lactating animals at the time of the antigenic transition from “juvenile” to “adult” type. The “adult” stage parasitemia in suckling pups was selectively reduced when compared to that of nonlactating adult rats. The lactating rat serum factor could be passively transferred with lactating rat serum to animals already weaned.     

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.     

Prolongation of skin xenograft survival with modified cultured fibroblasts

Cyclosporine A, one of the most potent immunosuppressive drugs, mediates some of its immunosuppressive and nephrotoxic effects by enhancing transforming growth factor-beta secretion and receptor expression. In this experimental study, the effect of cyclosporine pretreatment of cultured dermal fibroblasts on xenogeneic tissue rejection after microimplantation beneath skin grafts was investigated. The effects of the site-specific immunosuppressive strategy on skin xenograft survival were tested. Because the skin is an immunological indicator of the rejection of composite tissue allografts, it was considered that this strategy could be used as a supportive therapy for composite tissue allotransplantation in the future. In the first stage of the study, fibroblast cultures obtained from skin biopsy samples from five rats were treated with different single doses of cyclosporine (100 to 3000 ng/ml), and transforming growth factor-beta levels were measured in culture supernatants after 72 hours. In the second stage, 60 Sprague-Dawley rats were divided into six groups, as follows. For group I (sham), only the standard grafting procedure was performed. For group II, after the standard grafting procedure, rats were treated with intraperitoneal injections of 30 mg/kg (n = 5) or 10 mg/kg (n = 5) cyclosporine for 10 days. For group III, cultured fibroblasts obtained from skin biopsy samples from rats were treated with 100 or 500 ng/ml cyclosporine, and the cells were collected by light trypsinization and centrifugation after 72 hours. After the standard skin grafting procedure, modified fibroblasts were implanted between the graft and the recipient bed with a Pasteur pipet. For group IV, the same procedures as for group III were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. For group V, in addition to standard grafting, unmodified fibroblasts (not treated with cyclosporine) were implanted between the graft and the recipient bed. For group VI, the same procedures as for group V were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. The rejection process was observed macroscopically, and statistical significance was determined with the Mann-Whitney test (p < 0.01). Graft survival times were significantly prolonged in groups III and IV, compared with groups I, II, V, and VI (p < 0.001). No difference between groups III and IV was observed. The novel finding of this investigation is that xenogeneic skin graft survival times could be prolonged with microimplantation of cyclosporine-treated cultured fibro-blasts.     

Effect of localized cytokine dysregulation: accelerated rejection of IL-2-expressing skin grafts

Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site.     

VSD负压引流技术结合游离植皮治疗大面积皮肤缺损的 临床疗效研究

目的：研究VSD负压引流技术结合游离植皮治疗大面积皮肤缺损临床疗效。方法：选择2012年4月至2013年10月入我院接受治疗的大面积皮肤缺损患者65例,将其随机分为观察组（35例）与对照组（30例）。观察组患者术前先采用VSD治疗,游离植皮术后继续给予VSD治疗;对照组患者采取打压植皮或传统打包加压治疗。观察和比较两组患者的皮片成活时间、皮片成活率、术后住院治疗时间、换药次数、疼痛评分及家属满意度。结果：观察组患者的皮片成活时间短于对照组、皮片成活率高于对照组、住院时间短于对照组,换药次数少于对照组,两组之间的差异具有统计学意义（P〈0．05）。此外,观察组患者的疼痛视觉模拟评分明显低于对照组,患者家属的满意度评分明显高于对照组,差异具有统计学意义（P〈0．05）。结论：相对于传统的打压植皮或传统打包加压的技术而言,VSD负压引流技术结合游离植皮在治疗大面积皮肤缺损的临床疗效更好,值得在临床上推广使用。     

Host response to tissues placed in the anterior chamber of the eye: demonstration of migration inhibition factor and serum blocking activity.

Allograft immunity of rats to transplantation antigens was demonstrated by in vitro migration inhibition procedures. Spleen cell suspensions from rats sensitized to histocompatibility antigens by skin graft or anterior chamber implants failed to migrate normally in vitro when incubated in the presence of appropriate donor tissue extracts. Tissue grafts transplanted across both major and minor histocompatibility barriers had prolonged survival within the anterior chamber. However, 2 weeks after implantation, the recipient rats showed detectable sensitization to their implants. Serum from 14- to 32-day implant-bearing rats blocked the migration inhibition found in the implanted rats in the presence of corresponding tissue antigen. Such blocking activity was undetectable in the serum from rats which had implants for 7 days or after rejection of the implant was evident. MIF production in implanted and skin-grafted rats was evident at significantly different times in relation to the graft rejection. The asynchrony of MIF synthesis observed in these experimental animals leads us to postulate that the graft rejection and MIF production may be mediated by distinct lymphocyte populations. In addition, the route of the antigen presentation may account in part for the observed differences.     

Prolonged survival of skin allografts in rats by injection of serum or a serum fraction extracted from pregnant rats

This work report the partial purification of an alpha 2-macroglobulin present in the serum of allopregnant rats. Ultrogel filtration followed by immune absorption on different antisera were used giving a single band on polyacrylamide gel electrophoresis. This fraction was able to inhibit in vitro T cell cytotoxic response to allogenic cells. When administered repeatedly to rats it significantly increased the survival of skin allograft. This alpha 2-macroglobulin fraction isolated from allopregnant rats was denominated IRG to its graft rejection inhibitory activity.     

The prostacyclin analogue taprostene and recombinant human superoxide dismutase increase the permanent survival rate of endotoxemic rats

The permanent survival rate of endotoxemic rats was studied. In 40 untreated control rats the survival rate 7 days after the endotoxin administration was 33%. The survival rate was increased to 40% with recombinant human superoxide dismutase (r-HSOD), to 55% (p less than 0.05) with the prostacyclin analogue Taprostene and to 70% (p less than 0.01) with the combination of r-HSOD and Taprostene (20 rats for each treatment group). r-HSOD and Taprostene provide significant and permanent protection in rat endotoxic shock.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Detection of early pregnancy factor in swine: A need for dialogue

The rosette inhibition test, which has been used to detect early pregnancy factor (EPF) and other immunosuppressive factors in the serum of pregnant mice, women, and sheep, was adapted for use in swine. Since the initial methods described for use of the assay were cumbersome and time consuming, our first efforts were to simplify the technique. Substitution of microtiter plates for test tubes and treatment of sheep erythrocytes with AET to increase numbers and stability of the rosettes were successful. Although consistently higher rosette inhibition titers were obtained after incubation of lymphocytes in the serum of pregnant sows compared to controls, the variability in numbers of rosettes obtained on a day to day basis and the cumbersome nature of the test have impeded progress in firmly establishing the presence of EPF in pregnant swine and demonstrating the homology between it and the EPF reported in other species.     

Identification of molecules involved in the 'early pregnancy factor' phenomenon.

An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.     

Identification of a putative inhibitor of early pregnancy factor in mice

Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.