Skin grafts from genetically modified α-1,3-galactosyltransferase knockout miniature swine: A functional equivalent to allografts.

Burn is associated with a considerable burden of morbidity worldwide. Early excision of burned tissue and skin grafting of the resultant wound has been established as a mainstay of modern burn therapy. However, in large burns, donor sites for autologous skin may be limited. Numerous alternatives, from cadaver skin to synthetic substitutes have been described, each with varying benefits and limitations. We previously proposed the use of genetically modified (alpha-1,3-galactosyl transferase knockout, GalT-KO) porcine skin as a viable skin alternative. In contrast to wild type porcine skin, which has been used as a biologic dressing following glutaraldehyde fixation, GalT-KO porcine skin is a viable graft, which is not susceptible to loss by hyperacute rejection, and undergoes graft take and healing, prior to eventual rejection, comparable to cadaver allogeneic skin. In the current study we aimed to perform a detailed functional analysis of GalT-KO skin grafts in comparison to allogeneic grafts for temporary closure of full thickness wounds using our baboon dorsum wound model. Grafts were assessed by measurement of fluid loss, wound infection rate, and take, and healed appearance, of secondary autologous grafts following xenograft rejection. Comparison was also made between fresh and cryopreserved grafts. No statistically significant difference was identified between GalT-KO and allogeneic skin grafts in any of the assessed parameters, and graft take and function was not adversely effected by the freeze-thaw process. These data demonstrate that GalT-KO porcine grafts are functionally comparable to allogeneic skin grafts for temporary closure of full thickness wounds, and support their consideration as an alternative to cadaver allogeneic skin in the emergency management of large burns.

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts.

The emergence of skin-containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin-resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II-mismatched chimeras were tolerant of VCAs. MHC class I-mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient-type CD8+ lymphocytes. Systemic donor-specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Delayed type IV hypersensitivity reaction to porcine acellular dermal matrix masquerading as infection resulting in multiple debridements.

INTRODUCTION: Delayed type IV hypersensitivity reactions are well established in the surgical setting with respect to external exposure via topical antibiotics and internal exposure via synthetic materials. In contrast, biologic matrix is derived from decellularized human or animal tissues and is consequently believed to elicit a minimal host inflammatory response. OBJECTIVE: We report a case of delayed type IV hypersensitivity reaction secondary to a biologic comprised of porcine-derived acellular dermal matrix, [Strattice™]. CONCLUSIONS: While biologic matrix is often preferred over synthetic mesh due to its decreased risk for infection, this case emphasizes that potential for hypersensitivity to the material persists. Type IV hypersensitivity reactions should be included in the differential diagnosis for suspected post-operative infections.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Vascularized composite allograft tolerance across MHC barriers in a large animal model.

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

Wharton's Jelly stem cells: future clinical applications.

This review focuses on the therapeutic potential of stem cells harvested from the Wharton's Jelly of the human umbilical cord. Recently, investigators have found that a potent stem cell population exists within the Wharton's Jelly. In this review, the authors define a new subset of stem cells, termed perinatal stem cells, and compare them to other sources of stem cells. Furthermore, cryopreservation of Wharton's Jelly stem cells is described for potential use in future cell based therapies and/or regenerative medicine applications. Current evidence of the application of mesenchymal stem cells from various sources in both pre-clinical and clinical trials is reviewed in the context of potential indications of use for Wharton's Jelly derived mesenchymal stem cells.

Human umbilical cord blood-derived mast cells: a unique model for the study of neuro-immuno-endocrine interactions.

Findings obtained using animal models have often failed to reflect the processes involved in human disease. Moreover, human cultured cells do not necessarily function as their actual tissue counterparts. Therefore, there is great demand for sources of human progenitor cells that may be directed to acquire specific tissue characteristics and be available in sufficient quantities to carry out functional and pharmacological studies. Acase in point is the mast cell, well known for its involvement in allergic reactions, but also implicated in inflammatory diseases. Mast cells can be activated by allergens, anaphylatoxins, immunoglobulin-free light chains, superantigens, neuropeptides, and cytokines, leading to selective release of mediators. These could be involved in many inflammatory diseases, such as asthma and atopic dermatitis, which worsen by stress, through activation by local release of corticotropin-releasing hormone or related peptides. Umbilical cord blood and cord matrix-derived mast cell progenitors can be separated magnetically and grown in the presence of stem cell factor, interleukin-6, interleukin-4, and other cytokines to yield distinct mast cell populations. The recent use of live cell array, with its ability to study such interactions rapidly at the single-cell level, provides unique new opportunities for fast output screening of mast cell triggers and inhibitors.

Laser blepharoplasty with transconjunctival orbicularis muscle/septum tightening and periocular skin resurfacing: a safe and advantageous technique.

Carbon dioxide (CO2) laser blepharoplasty with orbicularis oculi muscle tightening and periorbital skin resurfacing is a safe procedure that produces excellent aesthetic results and diminishes the occurrence of complications associated with skin and muscle resection in the lower lid, particularly permanent scleral show and ectropion. The authors present a review of 196 cases of carbon dioxide laser blepharoplasty and periocular laser skin resurfacing performed at their center from April of 1994 to September of 1998. Of these cases, 113 patients underwent four-lid blepharoplasty, 59 underwent upper lid blepharoplasty only, and 24 underwent lower lid blepharoplasty only. Prophylactic lateral canthopexy was performed in 24 patients. Concomitant procedures (brow lift/rhytidectomy/rhinoplasty) were performed in 92 patients. The carbon dioxide laser blepharoplasty procedure resulted in no injuries to the globe, cornea, or eyelashes. Combined with laser tightening of the orbicularis oculi muscle and septum and periocular skin resurfacing, the transconjunctival approach to lower blepharoplasty preserves lower lid skin and muscle. Elimination of the traditional scalpel skin/muscle flap procedure results in a dramatically lower complication rate, particularly with regard to permanent ectropion and scleral show. Laser shrinkage of the orbicularis muscle and septum through the transconjunctival incision enables the correction of muscle aging changes such as orbicularis hypertrophy and malar festoons. The addition of periocular resurfacing enables the correction of skin aging changes of the eyelid that are not addressed by traditional scalpel blepharoplasty. In addition, lateral canthopexy constitutes an important adjunct to the laser blepharoplasty procedure for the correction of lower lid canthal laxity.

Tubulation repair of peripheral nerves in the rat using an inside-out intestine sleeve.

This study compared the regeneration of peripheral nerves in the Sprague-Dawley rat through a nerve guide prepared from rat small intestine to nerve regeneration using a standard autogenous nerve-graft repair strategy. In one experimental group (n = 15), inside-out rat intestine sleeves were used as nerve guides to bridge a 10-mm defect created in the right sciatic nerve. These nerve guides were prepared by harvesting 14-mm segments of small intestine from Sprague-Dawley rats not otherwise used in the study. The segments were turned inside-out to expose the serosa as the lumen of the guide, and transected nerve stumps were secured 2 mm into the guide on each end with an epineural-to-guide stitch. The control group (n = 15) had an identical gap repaired with a standard autologous nerve graft. Five animals from each group were sacrificed at 4, 8, and 12 weeks. The extent of axonal regeneration was assessed by axon-counting, retrograde tracer analysis, electromyography, and qualitative histologic assessment. The inside-out intestine sleeve group exhibited faster conduction velocities and greater axon counts when compared to the autologous nerve-graft controls. These novel nerve guides proved simple to manufacture and were completely absorbed by 12 weeks.

Suction-assisted lipectomy for lipodystrophy syndromes attributed to HIV-protease inhibitor use.

The addition of HIV-protease inhibitors to the arsenal of therapies for the treatment of HIV infection has resulted in significant suppression of viral load such that HIV-positive individuals experience reduced morbidity and extended life expectancy. Recently, a number of syndromes have been described involving abnormal fat distribution that may be associated with prolonged HIV-protease inhibitor therapy. These syndromes include hypertrophy of the cervicodorsal fat pad ("buffalo hump"); a tendency toward increased central adiposity ("protease paunch"); adiposity in the submental, mandibular, and lateral cheek regions of the face; and hypertrophy of adipose tissue in the breast in women. A peripheral lipodystrophy, or fat-wasting, in the extremities and face (particularly the malar and nasolabial fold regions) has also been observed. As these patients live longer and healthier lives, many are beginning to seek surgical correction of the disfigurements. In this regard, we present a review of the literature regarding these recently described syndromes to familiarize plastic and reconstructive surgeons with the unique deformities encountered in this ever-increasing patient population. We also present our results with suction-assisted lipectomy for treatment of these deformities. Physical findings, pathogenesis, and surgical management are discussed.

Hyaluronic acid enhances peripheral nerve regeneration in vivo.

Hyaluronic acid has been shown to enhance peripheral nerve regeneration in vitro. It has been proposed that, during the fibrin matrix phase of regeneration, hyaluronic acid organizes the extracellular matrix into a hydrated open lattice, thereby facilitating migration of the regenerating axons. Hyaluronic acid solutions and saline control solutions were injected into a nerve guide spanning a transected gap in the sciatic nerve of Sprague-Dawley rats (five in each group). Nerve conduction velocities were measured at 4 weeks by electromyography (EMG) before sacrifice of the animals. These studies demonstrated increased conduction velocities in the hyaluronic acid group compared with control animals (P = 0.006). After the animals were sacrificed, regenerated axon cables were quantified histologically, and axon branching was delineated by retrograde tracer analysis. In addition, the hyaluronic acid group showed an increase in myelinated axon counts at 4 weeks (P= 0.03). An increase in retrograde flow was demonstrated in the hyaluronic acid groups compared with animals receiving saline solution.

Collection and cryopreservation of cord blood for the treatment of hematopoietic disorders: the obstetrician's overview.

Bone marrow transplantation provides successful treatment for many diseases of the immune and hematopoietic systems. The main therapeutic ingredient in this procedure is stem cells collected from the bone marrow. Recently, it has been demonstrated that stem cells from human umbilical cord blood can serve as an alternative to bone marrow transplantation in children. Although cord blood transplantation in adults has not yet been attempted, it appears that there are enough stem cells present in cord blood for successful engraftment in adults. Obstetric health care providers should be aware that many familial conditions are treatable by cord blood stem cell transplantation in children. Obstetric health care providers caring for patients with familial disorders should consider counseling such patients regarding the collection and storage of cord blood for potential future uses in autologous or allogeneic transplantation.

Twin transfusion syndrome: reassessment of ultrasound diagnosis.

OBJECTIVE: To assess the antenatal ultrasonic diagnosis of the twin transfusion syndrome, an enigmatic disorder that results in an imbalance in the blood flow between monochorionic twins. STUDY DESIGN: Retrospective review of 16 cases of twin transfusion syndrome confirmed by placental pathology and neonatal data. RESULTS: A disparity in antenatal fetal weights, size difference between the two amniotic sacs and a single placenta were present in 81% (13/16) of confirmed cases. The finding of two separate umbilical cords with a disparity in the size or number of vessels was rare. Evidence of hydrops in either fetus or findings of congestive cardiac failure in the recipient twin were uncommon. CONCLUSION: Our data strongly suggest that the twin transfusion syndrome can be diagnosed in a significant number (81%) of cases. The syndrome is variable, and it is rare to observe all the diagnostic criteria in one case.

Double-blind; placebo-controlled trial of piperacillin prophylaxis in preterm membrane rupture.

OBJECTIVE: We attempted to test whether antibiotic therapy prolongs pregnancy in preterm premature rupture of membranes, because preterm premature rupture of membranes is frequently associated with chorionic-decidual infection. STUDY DESIGN: Women with preterm premature rupture of membranes and a singleton gestation at 24 to 34 completed weeks were randomized to receive either piperacillin 3 gm or placebo intravenously every 6 hours for 72 hours and were managed conservatively until spontaneous delivery, chorioamnionitis, or fetal distress. RESULTS: Between January 1987 and January 1992, a total of 75 patients were randomized to receive piperacillin (n = 38) or placebo (n = 37). There were no differences between the piperacillin group and the placebo group in mean gestational age at randomization (30.2 +/- 3 vs 30.3 +/- 2.9 weeks). However, a greater number of patients had pregnancy prolonged beyond 7 days (42.1% vs 10.8% p = 0.005) and the mean latency period was significantly prolonged (11.4 +/- 18.8 vs 6.1 +/- 13.6 days, p = 0.001) in the piperacillin group compared with the control groups. CONCLUSIONS: Use of intravenous piperacillin for 72 hours in preterm premature rupture of membranes significantly prolongs the latency period between membrane rupture and delivery.

Indomethacin in severe twin-to-twin transfusion syndrome.

Despite advances in maternal fetal medicine, the management of severe twin-to-twin transfusion syndrome in the second trimester presents a significant challenge. Presently, there is no uniformly accepted management protocol that is available for the treatment of this syndrome. We report the use of indomethacin in three cases of severe twin-to-twin transfusion syndrome in the second trimester. In the three cases of severe twin-to-twin transfusion syndrome no reduction of amniotic fluid in either sac was demonstrated. Two cases were complicated by single intrauterine fetal death within 72 hours of initiating indomethacin therapy. Because of our experience with these three cases, we conclude that indomethacin does not prevent perinatal mortality in patients with severe twin-to-twin transfusion syndrome.

A rapid visual test for predicting fetal lung maturity.

A rapid bedside test has been devised that enables an untrained observer to predict (p less than 0.001) when amniotic fluid will be greater than or equal to 0.15 at an optical density of 650 nm, lecithin/sphingomyelin ratio will be greater than or equal to 2.0, or when phosphatidylglycerol will be present. By a visual comparison of the turbidity of unspun amniotic fluid against positive (mature) or negative (immature) controls, technicians and resident physicians who had had no special training were able to classify correctly 87.2% (82/94) of unknown amniotic fluid samples. The sensitivity of the new test is 90.8% (58/65); the specificity is 70.3% (23/29). Thus, when more sophisticated methods are not readily available, we believe that this easily performed and accurate test can provide supplemental or preliminary data for patient management. In remote geographic areas our method could serve as the primary source of information about fetal lung maturity.

Antepartum management of twin gestation.

Twin gestation is now an area of vital concern to the perinatologist. Within the last decade a substantial reduction in perinatal mortality has been achieved, largely through advances in neonatal intensive care. However, preterm birth and its consequences remain the most important causes of perinatal morbidity and mortality. Twin pregnancy accounts for approximately 10% of all premature deliveries. From the data currently available, there is no justification for the routine use of hospital bedrest, prophylactic tocolytic agents, or elective cerclage in the management of twin pregnancy. Programs directed at the prevention of perterm delivery in twin pregnancy will be the focus of more scientific research. The use of ultrasound and antepartum testing in the form of the nonstress test, the biophysical profile, and Doppler ultrasound may lead to major advances in the diagnosis of intrauterine growth retardation and the twin-to-twin transfusion syndrome in twins. Therefore, it is reasonable to expect that fetal death and neonatal death from intrauterine growth retardation can be prevented.

The legal liability of the medical consultant in pregnancy.

The potential for professional liability that accompanies complications associated with obstetric care can be minimized by an understanding of the roles and responsibilities of both the consultant and the obstetrician. Critical to this is a fundamental knowledge of the medical malpractice action from a legal perspective. This article discusses several issues involved in professional liability as it relates to the obstetrician/gynecologist.

Bulimia nervosa in pregnancy: a case report.

Bulimia nervosa is an eating disorder characterized by secretive binge eating and purging with induced vomiting, laxatives, and diuretics. The disorder primarily afflicts young white women between 18-35 years of age. We report the case of a 30-year-old pregnant woman with a 17-year history of bulimia that involved up to six episodes of binging and purging daily. A multidisciplinary approach enabled outpatient management throughout gestation. The pregnancy resulted in the delivery of a normal 3000-g female infant at term. The ramifications for maternal and fetal well-being and the goals of therapeutic management are discussed.