Functional characteristics of a Ficoll-separated mouse bone marrow cell population involved in skin allograft prolongation

Treatment of recipient mice with donor bone marrow cells and anti-lymphocyte serum (ALS) results in extensive skin graft prolongation beyond that achieved in animals given only ALS. In this study, B6AF1 recipient mice were grafted with C3H/He skin on day 0, treated with ALS on days -1 and +2 and infused on day +7 with donor strain (C3H/He) bone marrow cells. Extensive graft prolongation was achieved either with 25 X 10(6) whole bone marrow cells or with 1 X 10(6) lymphoid-like cells derived from donor marrow that sediment at a rate of 3 mm/hr in a 2 to 4% Ficoll gradient at unit gravity. These allograft-prolonging lymphoid-like cells appear not to be CFUs cells, have suppressive activity in in vitro MLC assays, and contain both nylon wool adherent and non-adherent cells. These studies thus show that allograft promoting cells can be isolated from bone marrow utilizing Ficoll gradients. Functional studies suggest that 3 mm/hr sedimenting donor bone marrow suppressor cells may be involved in the induction of allograft prolongation.     

Regulatory roles of NKT cells in the induction and maintenance of cyclophosphamide-induced tolerance

We have previously reported the sequential mechanisms of cyclophosphamide (CP)-induced tolerance. Permanent acceptance of donor skin graft is readily induced in the MHC-matched and minor Ag-mismatched recipients after treatment with donor spleen cells and CP. In the present study, we have elucidated the roles of NKT cells in CP-induced skin allograft tolerance. BALB/c AnNCrj (H-2(d), Lyt-1.2, and Mls-1(b)) wild-type (WT) mice or Valpha14 NKT knockout (KO) (BALB/c) mice were used as recipients, and DBA/2 NCrj (H-2(d), Lyt-1.1, and Mls-1(a)) mice were used as donors. Recipient mice were primed with 1 x 10(8) donor SC i.v. on day 0, followed by 200 mg/kg CP i.p. on day 2. Donor mixed chimerism and permanent acceptance of donor skin allografts were observed in the WT recipients. However, donor skin allografts were rejected in NKT KO recipient mice. In addition, the donor reactive Vbeta6(+) T cells were observed in the thymus of a NKT KO recipient. Reconstruction of NKT cells from WT mice restored the acceptance of donor skin allografts. In addition, donor grafts were partially accepted in the thymectomized NKT KO recipient mice. Furthermore, the tolerogen-specific suppressor cell was observed in thymectomized NKT KO recipient mice, suggesting the generation of regulatory T cells in the absence of NTK cells. Our results suggest that NKT cells are essential for CP-induced tolerance and may have a role in the establishment of mixed chimerism, resulting in clonal deletion of donor-reactive T cells in the recipient thymus.     

Cytogenetic studies using Q-band polymorphisms in patients with AML receiving marrow from like-sex donors

Summary After bone marrow transplantation (BMT), it is important to monitor the bone marrow and lymphoid cell populations of the recipient to document engraftment. When donor and recipient are of unlike sex, the sex chromosomes serve as a useful marker to determine cellular origin. When donor and recipient are of like sex, autosomal heteromorphisms can be used to identify the origin of cells in metaphase. Using Q-banding, we found that 17 of 20 patient/donor pairs (85%) examined showed at least one chromosome heteromorphism that distinguished between recipient and donor cells with certainty. Five of the patients were followed up after BMT in order to document engraftment. Donor metaphases could be detected in the marrow within two weeks of BMT when the graft was successful. Chimaerism was detected in the lymphocyte population even when the graft persisted. In a case of graft failure, donor cells did not persist in the marrow, and the lymphocyte population did not convert to donor type. These studies demonstrate that autosomal heteromorphisms are useful in the study of myeloid and lymphoid chimaeric states after BMT.     

A simple method of preparing split-thickness skin grafts for meshing

A simple and rapid technique of mounting a split-thickness skin graft on the Tanner dermacarrier is presented. Initially, petroleum jelly is applied onto the grooved surface of the dermacarrier. The skin graft is not removed from the donor site; instead, the skin graft knife is withdrawn backwards, allowing the graft to slip back, smoothly and evenly, onto the donor site through the knife. The dermacarrier, with the petroleum jelly, is laid onto the graft. The graft adheres to the dermacarrier because of the jelly. No further maneuver is necessary prior to meshing. With this technique it is impossible to apply the graft cut-side up on the recipient site.     

Inhibition of terminal complement components in presensitized transplant recipients prevents antibody-mediated rejection leading to long-term graft survival and accommodation

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.     

Energetics of mice in stable and unstable social conditions: Evidence of an air-borne factor affecting metabolism

The metabolic rates of laboratory mice were compared in three conditions: isolated mice, mice paired together over six days (stable groups), and mice paired with strange partners daily (unstable groups). Stable pairs had 15% lower metabolic rates than either isolated or unstable pairs. In other experiments when two mice were placed in separate metabolic chambers and connected together via an air flow, the metabolic rate of the recipient in the series was 35% lower than the donor. The data suggest that a ‘factor’ produced by the donor mouse was passed via the air supply into the recipient's chamber.     

The effects of macrophage-mediated inflammatory response to the donor site on long-term retention of a fat graft in the recipient site in a mice model

Inflammatory responses mediated by macrophages play a role in tissue repair. However, it is unclear whether the repair in the donor site after liposuction would have any effects on fat graft retention in the recipient site. This study is designed to evaluate the effects of a macrophage-mediated inflammatory response in donor sites on long-term retention of fat grafting. In this study, mice were randomly divided into two groups. One underwent simulated liposuction, called the fat procurement plus grafting (Pro-Grafting) group, and the other underwent sham surgery, called the fat grafting only (Grafting Only) group. The prepared fat (0.3 ml each) was engrafted and cellular events over a 90-day period were assessed. We found macrophages were infiltrated into adipose tissue at the recipient site in the Grafting Only group within 7 days and the repair essentially completed within 30 days. By contrast, few macrophages infiltrated the recipient site in the Pro-Grafting group within 7 days and the entire remodeling process took 30 days longer in the Pro-Grafting than the Grafting Only group. Moreover, C-reactive protein levels were immediately upregulated after surgery, and the inflammatory factors' expression was higher at the donor rather than the recipient site. However, the repair processes and the long-term retention rate became normal when the adipose tissue was grafted after the donor site did not require macrophages for repair. Therefore, we suggest higher inflammatory factors promote macrophage infiltration and the adipose tissue regeneration process at the donor site. This process is delayed at the recipient site, which may affect long-term retention of fat grafts.     

Survival of skin allografts following embryonic limb bud transplants in the turtle,Chelydra serpentina

Allografts of skin were observed in Chelydra serpentina. The response to these grafts was modified by a previous transplantation of a limb bud at an early embryonic stage. When the same donor was used for all transplants, the first skin graft was accepted by the host. A second skin graft, however, was rejected at about the rate of a simple first set allograft of skin. The animals were conditioned by the embryonic limb graft; this embryonic graft can be undergoing rejection at the same time a first set skin graft from the same donor was being accepted. The tolerance induced by the embryonic graft was sepcific for its donor.     

The epidermis as a graft

Laboratory epidermal autotransplantation was performed on the surface of a full-thickness skin defect using mongrel female rats. Epidermal graft represented suction blister roofs, formed as the result of the donor skin site treatment with lowered up to -0.6 kg/cm2 pressure. It contained all epidermal cell layers. Following 1, 7 and 28 days after the transplantation recipient bed sites containing grafted epidermis were excised and histological study war performed. It was demonstrated that epidermal graft received by the method described was able to grow as well as to differentiate on the surface of a full-thickness skin defect.     

Perforin-dependent cell death in skin allograft rejection of the terrestrial slug Incilaria fruhstorferi

The rejection of allografts in mammals is mainly mediated by cytotoxic T-lymphocytes, whereas no comparable immunoreactive cells have been described in invertebrates. The present study was undertaken to determine whether similar cytotoxic effector cells are present when allograft rejection occurs in the terrestrial slug Incilaria fruhstorferi. A piece of dorsal skin from a donor animal was orthotopically transplanted to a recipient. Immunohistochemistry for perforin, detection of apoptosis by the TUNEL (TdT-mediated dUTP-biotin nick-end labeling) method, and electron microscopy were performed using both donor and recipient tissues. Cellular changes in the rejection process continued over for 40 days. Two functional types of "effector" cells were recognized at the rejection site, but they were observed to be macrophages possessing perforin granules and phagocytosing damaged cells of the allograft. Three days after transplantation, the perforin-positive cells were recognized only in the recipient tissue surrounding the allograft. Five days after transplantation, these cells started to appear in the graft, while they disappeared from the host tissue. However, TUNEL-positive cells were not observed throughout the graft-rejection process. Electron microscopic examination of the graft tissue revealed autophagic degeneration of epithelial cells, mucous cells, pigment cells, fibroblasts, and muscle cells. These observations suggest that the molluscan slug has the capability to recognize differences in cell-surface molecules between the allogeneic and recipient tissues, and that an allograft is chronically rejected due to a type of immunocyte that can induce perforin-dependent cell death.     

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.     

Latissimus dorsi free muscle flap in lower-extremity reconstruction

The free latissimus dorsi skin-muscle flap has gained wide popularity to solve a variety of difficult reconstructive surgical problems. However, the donor site of this skin-muscle flap leaves a conspicuous scar and indentation, and frequently in the recipient site the skin-muscle flap leaves a conspicuous scar and indentation, and frequently in the recipient site the skin-muscle flap requires staged defatting procedures. This case demonstrates the use of the latissimus dorsi muscle flap for lower-extremity reconstruction, where a new blood supply and soft-tissue coverage are required to solve a chronically infected, open ankle joint. By taking the latissimus muscle only through a short, axillary incision, much of the donor-site deformity is minimized, and after transfer, the muscle can be molded and shaped to fit the recipient site with split-thickness skin graft coverage. This combination of free muscle flap transfer and skin graft would appear to provide a flexible, contoured, well-vascularized muscle with a relatively inconspicuous incision and skin-graft donor site.     

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.     

Long-term survival of human skin allografts in patients with immunosuppression

Severe burn patients lack adequate skin donor sites to resurface their burn wounds. Patients with severe burn injuries to areas such as an entire face are presently reconstructed with skin grafts that are inferior to normal facial skin. This study was designed in part to determine whether human skin allografts would survive, repopulate, and persist on patients with immunosuppression and after discontinuation of immunosuppression. Small split-thickness skin grafts were synchronously transplanted at the time of renal transplantation from six renal transplant donors to recipients. All six patients were immunosuppressed with the usual doses of renal transplant immunosuppressants (methylprednisolone, cyclosporine, prednisone, and azathioprine). The skin allografts were biopsied when rejection was suspected and at various intervals. Special histologic studies were performed on skin biopsy specimens. Class II DNA tissue typing was performed on transplanted and autogenous skin biopsy specimens of four patients. Fluorescent in situ hybridization was performed successfully on skin biopsies of four patients' transplanted skin and on two of these four patients' autogenous skin. All six human skin allografts sustained a 100 percent take and long-term clinical survival. DNA tissue typing performed on skin allograft biopsy specimens from patients taking immunosuppressants all revealed donor and recipient cells. DNA tissue typing performed on autogenous skin biopsies from the same patients all revealed only recipient cells. Fluorescent in situ hybridization performed on allograft and autogenous specimens from patients taking immunosuppressants revealed transplanted donor cells with rare recipient cells in the allograft and only recipient cells in the autogenous skin. This study of six patients proves that it is possible for human skin allografts to survive indefinitely on patients taking the usual dosages of immunosuppressants used for renal transplantation. There was minimal repopulation of skin allografts by autogenous keratinocytes and fibroblast while patients were taking immunosuppressants. Immunosuppression was discontinued in two patients after renal transplant rejection after 6 weeks and 5 years. When immunosuppression was discontinued after 5 years in one patient, the skin allograft cells were destroyed and replaced with autogenous cells, but the skin graft did not reject acutely and persisted clinically. It is hypothesized that the acellular portion of the skin allograft was not rejected acutely because of relatively low antigenicity and because it acted as a lattice for autogenous cells to migrate into and replace rejected allograft skin cells. No chimerism was seen in autogenous skin in the skin-renal transplant patients in this study.     

A systematic approach for improvement of the donor sites of fasciocutaneous flaps

Fasciocutaneous flaps as a group have been maligned more often for fear of potential donor-site morbidity than any concern for reliability. Typically, this is related to limitations imposed by the skin graft necessary to close most such donor sites, as admittedly has been required for the majority (52 percent) of our 313 flaps over the past 2 decades. Nevertheless, 48 percent did not require skin grafts, reflecting the adoption of strategies that evolved to minimize this shortcoming. These included use of fascia-only flaps, primary closure with small composite flaps, direct closure possible by use of rotation or advancement flaps or a second flap, or a delayed closure utilizing either pretransfer or posttransfer tissue expansion. Donor-site complications were actually fewest when a skin graft or primary closure was possible and occurred at the same rate regardless of body region. However, because the skin-grafted donor site was always a cosmetic compromise, a systematic approach to circumvent its use whenever possible is emphasized as a valuable tool to enhance the role of fasciocutaneous flaps as a vascularized flap alternative.     

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.     

Contribution of renal secreted complement C3 to the circulating pool in humans

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.     

Factors Associated with Mortality and Graft Failure in Liver Transplants: A Hierarchical Approach

Background

Liver transplantation has received increased attention in the medical field since the 1980s following the introduction of new immunosuppressants and improved surgical techniques. Currently, transplantation is the treatment of choice for patients with end-stage liver disease, and it has been expanded for other indications. Liver transplantation outcomes depend on donor factors, operating conditions, and the disease stage of the recipient. A retrospective cohort was studied to identify mortality and graft failure rates and their associated factors. All adult liver transplants performed in the state of São Paulo, Brazil, between 2006 and 2012 were studied.

Methods and Findings

A hierarchical Poisson multiple regression model was used to analyze factors related to mortality and graft failure in liver transplants. A total of 2,666 patients, 18 years or older, (1,482 males; 1,184 females) were investigated. Outcome variables included mortality and graft failure rates, which were grouped into a single binary variable called negative outcome rate. Additionally, donor clinical, laboratory, intensive care, and organ characteristics and recipient clinical data were analyzed. The mortality rate was 16.2 per 100 person-years (py) (95% CI: 15.1–17.3), and the graft failure rate was 1.8 per 100 py (95% CI: 1.5–2.2). Thus, the negative outcome rate was 18.0 per 100 py (95% CI: 16.9–19.2). The best risk model demonstrated that recipient creatinine ≥ 2.11 mg/dl [RR = 1.80 (95% CI: 1.56–2.08)], total bilirubin ≥ 2.11 mg/dl [RR = 1.48 (95% CI: 1.27–1.72)], Na⁺ ≥ 141.01 mg/dl [RR = 1.70 (95% CI: 1.47–1.97)], RNI ≥ 2.71 [RR = 1.64 (95% CI: 1.41–1.90)], body surface ≥ 1.98 [RR = 0.81 (95% CI: 0.68–0.97)] and donor age ≥ 54 years [RR = 1.28 (95% CI: 1.11–1.48)], male gender [RR = 1.19(95% CI: 1.03–1.37)], dobutamine use [RR = 0.54 (95% CI: 0.36–0.82)] and intubation ≥ 6 days [RR = 1.16 (95% CI: 1.10–1.34)] affected the negative outcome rate.

Conclusions

The current study confirms that both donor and recipient characteristics must be considered in post-transplant outcomes and prognostic scores. Our data demonstrated that recipient characteristics have a greater impact on post-transplant outcomes than donor characteristics. This new concept makes liver transplant teams to rethink about the limits in a MELD allocation system, with many teams competing with each other. The results suggest that although we have some concerns about the donors features, the recipient factors were heaviest predictors for bad outcomes.     

In vitro development of yak (Bos grunniens) embryos generated by interspecies nuclear transfer

Interspecies cloning may be used as an effective method to conserve highly endangered species, but at present it suffers from relatively low levels of efficiency. In order to find a technique that could be used in conservation of the wild yak (Bos grunniens), we designed in six separate experiments to investigate the following factors that might influence the efficiency of interspecies cloning: (1) maturation rates of the recipient bovine oocytes; (2) nuclear donor cell types; (3) age of the yak from which the yak ear skin fibroblast cell line originated; (4) donor cells treated with or without serum starvation; (5) nuclear donor gained from fresh cells or frozen-thawed cells; (6) effect of 0.5 or 1.5 h from fusion to activation. The results of experiment 1 showed that when recipient oocytes in a replicate had a maturation rate of <40% (34+/-3.0%; three replicates) the proportion of nuclear transferred oocytes that developed to blastocyst was 2+/-1.1%, which was significantly lower (P<0.01) than the 25+/-3.2% achieved when the recipient oocyte maturation rate was 71+/-3.7% (three replicates). The efficiency of blastocyst production was increased substantially (P<0.05) when the time from fusion to activation increased from 0.5 h (21+/-2.3%; three replicates) to 1.5 h (35+/-3.5%; five replicates; experiment 6). There was no significant effect of the source of the donor nuclei (ear skin fibroblast or cumulus cells), the age of the animal (3 months or 4 years) from which the donor cells were derived, serum deprivation of the donor cells, or the use of fresh or frozen-thawed donor cells (experiments 2-5). Transfer of three interspecies cloned blastocysts to each of 108 bovine recipients resulted in two pregnancies being established that did not survive to day 120 of gestation.     

Distally based lateral supramalleolar adipofascial flap for reconstruction of the dorsum of the foot and ankle

Soft-tissue reconstruction of the dorsum of the foot and ankle has long been a challenge for reconstructive surgeons. Limitations in the available local tissue and donor-site morbidity restrict the options. In an effort to solve these difficult problems, the authors have begun to use a distally based lateral supramalleolar adipofascial flap. This report presents the authors' early experience with seven patients treated with this flap. The patients' ages ranged from 5 to 26 years; four of the patients were male and three were female. The cause of the soft-tissue defects involved acute trauma and chronic scar contracture. The flap and the adjoining raw area were covered with a full-thickness skin graft, and the donor site at the lateral aspect of the leg was closed primarily without grafting. A skin graft was taken from the groin area, which was closed primarily. Compared with the other flaps, this adipofascial flap was thinner and produced less bulkiness to the recipient site and minor aesthetic sequelae to the donor site. It is believed that this flap is versatile and effective and is a good addition to the available techniques used by reconstructive surgeons for coverage of the dorsum of the foot and ankle.