Efficacy of RS-61443 in reversing acute rejection in a rat model of hindlimb allotransplantation.

The requirement for effective, minimally toxic immunosuppression remains a major obstacle to performing human composite tissue allotransplantation. The skin component of composite tissue (e.g., limb) allografts is especially antigenic, necessitating toxic immunosuppressant doses to prevent or reverse acute rejection. In previous experiments, RS-61443, an experimental mycophenolic acid ester that inhibits lymphocyte proliferation with minimal toxicity, prevented acute limb allograft rejection in rats for more than 8 months when started on the day of transplantation. In this study, the ability of RS-61443 to reverse established acute rejection was tested in a rat model of hindlimb allotransplantation. Brown-Norway donors and Fischer 344 recipients provided a MHC mismatch for orthotopic midfemur limb transplants that were performed with microsurgical repair of femoral vessels and sciatic nerve. Three groups were studied: untreated allografts (n = 6); allografts receiving RS-61443 at 30 mg/kg/day, started on postoperative day 7 (n = 11); and allografts receiving RS-61443 at 30 mg/kg/day, started on postoperative day 9 (n = 9). Skin and soft tissues were biopsied periodically to assess rejection. Untreated allografts had complete acute rejection within 12-13 days. Animals in both the 7- and 9-day groups developed moderate to severe rejection clinically and histologically before initiation of immunosuppressive therapy. In both groups, RS-61443 was able to reverse rejection completely in all animals from which biopsies were obtained at the time of death at 9-16 weeks after transplantation (P < 0.007). RS-61443 was highly effective as a primary immunosuppressant for reversing established acute rejection in rat hindlimb allografts.

A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443.

Although technically possible, limb allotransplantation has not been applied clinically. The skin component is especially antigenic, requiring high immunosuppressant doses with an unacceptable toxicity profile. RS-61443, an experimental mycophenolic acid ester that inhibits lymphocyte proliferation without major systemic toxicity, was tested as an immunosuppressant to prevent rejection of rat hindlimb allotransplants. Utilizing Brown-Norway donors and F344 recipients to provide a major mismatch at the MHC, midfemur orthotopic limb transfer was performed with microsurgical repair of femoral vessels and sciatic nerve. Four primary groups were studied: autografts (n = 4); untreated allografts (n = 6); allografts receiving CsA 10 mg/kg for 20 days, then twice per week (n = 6); and allografts receiving RS-61443 30 mg/kg/day (n = 6). Skin and soft tissues were biopsied to assess rejection. Autografts had indefinite limb survival, while untreated allografts had complete acute rejection within 10-12 days. Five of the six CsA rats developed delayed mild-moderate acute rejection within 6 months. In contrast, 5 of the 6 RS-61443 rats had no rejection after at least 32 weeks, while the sixth rat developed only slight rejection on skin biopsy. All animals regained full sensation and partial functional return. RS-61443 is highly effective as a primary immunosuppressant for hindlimb allotransplantation. The disturbing moderate rejection observed in CsA animals, which was absent with RS-61443, may significantly hamper function of transplanted limbs.

Use of combination of low-dose cyclosporine and RS-61443 in a rat hindlimb model of composite tissue allotransplantation.

Despite technical feasibility, composite tissue allotransplantation has not been applied clinically because of immunosuppressive toxicity associated with these highly antigenic allografts. Combination immunosuppression therapy can help overcome this obstacle by allowing lower doses of individual drugs and minimizing toxicity. RS-61443 (mycophenolate mofetil), an effective immunosuppressant that inhibits lymphocyte proliferation, was tested at subtherapeutic doses in combination with cyclosporine (CsA) in a rat hindlimb allotransplantation model with a major antigenic mismatch at the MHC. Five groups were studied: untreated autograft controls (n=4), untreated allograft controls (n=6), allografts receiving low-dose CsA 1.5 mg/kg/day (n=11), allografts receiving low-dose RS-61443 15 mg/kg/day (n=17), and allografts receiving combination low-dose CsA 1.5 mg/kg/day + RS-61443 15 mg/kg/day (n=18). The autograft controls survived indefinitely, while untreated allograft control animals developed severe rejection within 12 days. Subtherapeutic CsA and RS-61443 monotherapy groups developed acute rejection in 64% and 100% of rats, respectively. In contrast, only 11% of rats receiving combination therapy with CsA + RS-61443 at these same subtherapeutic doses developed acute rejection (P < or = 0.0013). Bone marrow toxicity, manifested primarily by anemia and measured objectively by hematocrits, was reduced significantly (P=0.04) in animals receiving low-dose RS-61443 therapy when compared with high-dose controls. These results confirm that subtherapeutic RS-61443 + CsA combination therapy is efficacious in preventing rejection while minimizing toxicity.

Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers.

BACKGROUND: Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. METHODS: Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. RESULTS: Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. CONCLUSIONS: Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.

The dynamic properties of tissue oxygen in healing flaps.

Oxygen delivery to random pattern and musculocutaneous flaps was investigated in a canine model. Oxygen tension was measured in the proximal and distal portions of each flap and in adjacent normal skin by means of a recently developed technique. The effect of delay techniques on tissue oxygen tension was also examined with modifications of the same flap model. All measurements were made over a range of inspired oxygen concentrations (21% to 100%) both before operation and at intervals up to 15 days after operation. Tissue oxygen tensions were significantly higher in the musculocutaneous flaps than in random pattern flaps up to 6 days after operation. They were higher in the proximal portions than in the distal portions in each flap type. This difference was greater in the random pattern flap. Delay techniques prevented the early dramatic decrease in postoperative oxygen tension seen in random pattern flaps. Differences in the pattern of oxygen delivery to random pattern and musculocutaneous flaps may in part explain the greater reliability of musculocutaneous flaps when transposed in the presence of infection.

The recalcitrant perineal wound after rectal extirpation. Applications of muscle flap closure.

Perineal wounds developing after abdominoperineal resection result in chronic purulent drainage and intermittent episodes of sepsis and are generally unresponsive to conservative medical and surgical treatment. Thirteen consecutive patients (aged 27 to 74 years; mean, 48 years) who underwent débridement and immediate muscle flap closure of these wounds were analyzed to identify risk factors for delayed healing and to evaluate the effectiveness of muscle flap coverage. Three risk factors were identified: preoperative or postoperative radiation therapy, resection for recurrent carcinoma, and inflammatory bowel disease. A total of 19 muscle flaps (11 gracilis, five gluteal thigh, two gluteus maximus, and one rectus abdominis) were used to close these wounds. During an average 3.5-year follow-up, four (31%) minor complications and one (8%) recurrence were noted to occur. Muscle flaps provide safe, effective, single-stage procedures for the closure of chronic perineal wounds.

Biologic priming of neutrophils in subcutaneous wounds.

Migration of neutrophils from blood into tissue is a complex response by circulating cells to chemotactic stimulation. Previous studies of the functional changes induced by this process have produced variable results. We compared neutrophils isolated from blood and from subcutaneous wounds in rabbits using established assays for adherence, chemotaxis, superoxide anion production, and hydrogen peroxide production. No differences in adherence to biologic surfaces or chemotaxis toward activated plasma were found. However, our results confirm the observation that wound neutrophils are "primed" for increased production of oxygen radicals. Primed responses were observed for both soluble (formyl methionyl leucylphenylalanine, phorbol myristate acetate) and particulate (opsonized zymosan) stimulants. Priming was also observed for peritoneal exudate neutrophils. The data suggest that the process of extravascular migration includes priming of the superoxide generating system.

Flap selection as a determinant of success in pressure sore coverage.

OBJECTIVE: To establish a treatment algorithm for the long-term surgical management of pressure sores. DESIGN: Retrospective case series. SETTING: University-based teaching hospital. PATIENTS: From March 1979 to July 1995, 280 unselected pressure sore reconstructions (113 ischial, 94 sacral, and 73 trochanteric sores) were performed in 201 patients (130 men and 71 women; age range, 16-90 years; mean, 50 years). Of the patients, 90% had severe spinal cord injuries (paraplegia or quadriplegia). Forty-one percent of the wounds were chronic (present longer than 3 months). MAIN OUTCOME MEASURES: Length of stay, postoperative morbidity and mortality, and flap success (defined as a completely healed wound). RESULTS: Overall, 89% of the flaps healed primarily (ischium, 83% [94/113]; sacrum, 91% [86/94]; trochanter, 93% [68/73]). Three fourths of cases were treated in a single stage (debridement and reconstruction). The inferior gluteus maximus island flap (ischium) (94% [32/ 34]), the V-Y gluteus maximus advancement flap (sacrum) (97% [36/37]), and the tensor fascia lata flap (trochanter) (95% [42/44]) had the highest success rates. Flap success was not significantly affected by the size of the pressure sore or the number of previous flaps used. Postoperative hospital stays averaged 20 days. The overall complication rate was 28%, most commonly from a slight wound edge dehiscence. CONCLUSIONS: Flap selection and the appropriate short- and long-term sequence of flap use significantly improve success rates for pressure sore coverage. Reconstruction can be reliably performed in a single stage with a relatively short hospitalization.

An adverse wound environment activates leukocytes prematurely.

This study was designed to evaluate the effect of different wound environments on wound activation. Our wound model provided two distinct environments, a well vascularized musculocutaneous flap and a poorly perfused random-pattern flap, in miniature swine. Leukocytes were isolated and analyzed by the following three variables: surface and total cellular Mac-1 (CD11b/CD18), superoxide anion expression, and lactoferrin release. Leukocytes from the unfavorable, poorly oxygenated wound environment activate on entry into the wound. Leukocytes from the musculocutaneous flap wound are better able to respond to a maximal challenge with the phorbol ester, phorbol myristate acetate. These findings may account for the enhanced bactericidal actions of the musculocutaneous flap compared with the random-pattern flap observed clinically.

Superoxide production by wound neutrophils. Evidence for increased activity of the NADPH oxidase.

Oxygen radical secretion by neutrophils is potentiated or "primed" by extravascular migration into wounds. To define this change in responsiveness more precisely we measured superoxide production by blood and wound neutrophils from rabbits using formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate as agonists. In all experiments, the time- and dose-dependency of superoxide secretion were the same for blood and wound neutrophils. However, wound neutrophils produced significantly more superoxide. Furthermore, the cytochrome b component of the NADPH oxidase was found in greater quantities within wound neutrophils. We conclude that priming does little to alter the requirements for activating the NADPH oxidase but does significantly increase the velocity of superoxide generation. The data suggest that alterations in the assembly and function of the NADPH oxidase may contribute to enhanced superoxide secretion by wound neutrophils.

Microsurgical reconstruction of the midface.

OBJECTIVE: To establish a treatment algorithm for reconstructing complex midfacial defects. DESIGN: Retrospective case series. SETTING: University-based teaching hospital. PATIENTS: Thirty-one consecutive patients were treated from 1991 through 1995. The 18 males and 13 females were aged 15 to 90 years (mean age, 58 years). The cause of the defect included neoplasm (n = 27) and trauma (n = 4). Reconstruction consisted of 1 of 4 free flaps: rectus abdominis, radial forearm, fibula, or latissimus dorsi. Aesthetic and functional results were determined by patient questionnaires and physical examinations. MAIN OUTCOME MEASURES: Length of stay, postoperative morbidity and mortality, degree of aesthetic and functional restoration, and detection of tumor recurrence. RESULTS: Twenty-seven (87%) of the 31 patients underwent reconstruction with a single major procedure. All of the flaps survived. Postoperative hospital stays averaged 14 days. Late tumor recurrence occurred in 7 (23%) of the 31 patients and was promptly detected. Aesthetic and functional results were rated good or excellent in 77% (24/31) and 87% (27/31) of patients, respectively. Of the 20 patients who underwent alveolar ridge resection, 16 (80%) received dental rehabilitation, 44% of whom received osseointegrated implants into either a bone flap or remaining native bone. Osseointegrated implants were inset during the initial reconstruction 57% (4/7 patients) of the time. CONCLUSIONS: For complex midfacial defects, free-flap transfer can be performed with a high degree of success, restoring both appearance and function in most patients. The only instance in which bone is necessary to reconstruct the midface involves those areas in which osseointegrated implants are needed, ie, alveolar ridge (dental implant) and/or orbit (ocular prosthesis). In such cases, the fibula osteocutaneous free flap is the flap of choice. Otherwise, soft-tissue flaps are selected based on wound size.

Reconstruction of posterior neck and skull with vertical trapezius musculocutaneous flap.

The vertical trapezius musculocutaneous flap has been successfully utilized for reconstruction in 13 patients with complex posterior skull and neck defects. This flap based on its vascular pedicle, the descending branch of the transverse cervical artery, provides well-vascularized tissue for coverage of defects related to chronic osteomyelitis, tumor extirpation, osteoradionecrosis, and dehisced cervical laminectomy wounds. Emphasis on flap design, including the location of the skin island, allows adequate wound coverage, direct donor site closure, and muscle function preservation. With its large size and wide arc of rotation, the vertical trapezius musculocutaneous flap provides reliable coverage for posterior trunk, cervical, and skull defects.

Platysma musculocutaneous flap: clinical and anatomic considerations in head and neck reconstruction.

The platysma musculocutaneous flap was used in 14 patients for reconstruction of the oral lining and external skin coverage in the head and neck area. Complications occurred in five patients. Only one patient, however, required further surgery and this was for correction of a poor cosmetic result. The skin and subjacent muscle are supplied by a major vascular pedicle from the facial artery and vein and a minor pedicle from the transverse cervical artery and vein. Either pedicle can be used as the pivot for the flap's arc of rotation. The donor defect can be closed primarily and did not present any problems. The flap can be raised with both its motor and sensory supply intact. The thinness and pliability, color match, and proximity make this flap a useful adjunct in head and neck surgery.

Influence of radiotherapy on microvascular reconstruction in the head and neck region.

Over a 5-year period at the University of California San Francisco, 42 patients who required microvascular reconstruction for abnormalities in the head and neck area were identified. Twenty-four patients (Group I) underwent reconstruction for a variety of neoplastic and non-neoplastic conditions and did not receive radiotherapy. Eighteen patients (Group II) had undergone previous radiotherapy averaging 6,090 rads. The mean ages for Group I and II patients were 40.7 and 55.5 years, respectively. In Group I, 13 muscle, 7 fasciocutaneous, 4 osteocutaneous, and 2 jejunal transfers were performed. In Group II, 11 muscle, 5 fasciocutaneous, 3 osteocutaneous, and 2 jejunal transfers were performed. Flap survival at 3 months was 88% in Group I and 95% in Group II. Wound complication rates were similar in both groups (15% Group I, 19% Group II), as was donor site morbidity (15% Group I, 29% Group II). Operative times (10.9 hours Group I, 10.6 hours Group II) and median postoperative hospitalization (14 days Group I, 16 days Group II) were comparable as well. Four of the five patients in whom the flap procedures failed were subsequently treated by a second microvascular reconstruction. Previous irradiation of the recipient bed did not appear to affect the success of subsequent microvascular reconstruction or the difficulty of such reconstruction as judged by operative time.

Reconstruction of the breast: where do we fall short? An evolution of ideas.

We have analyzed our 5 year experience with 153 breast reconstructions. There were 83 latissimus dorsi reconstructions, 16 simple silicone implant insertions, 21 transverse rectus abdominus myocutaneous flaps, and 33 reconstructions using a variety of other methods. The rate of postoperative complications was 24 percent. The most common shortcoming was the inability to obtain symmetry with the other breast. Persistent problems included inability to fill the subclavicular hollow or the superior con-cavity due to partial atrophy of the pectoralis major muscle, and particularly, the lack of projection and ptosis in the reconstructed breast.

Radiation injury.

Despite advance radiotherapy techniques, long-term complications of radiation injury are still commonly seen. Acute effects are largely time dependent and can be controlled by alteration of therapy schedule. Chronic effects are dose dependent, and are associated with increased fibrosis and decreased vascularity which can lead to tissue necrosis, infection, and ulceration. The damaging effects of radiation therapy may not be clinically apparent for months or even years after treatment is given. The most commonly accepted theory explaining the effects of radiation injury on tissue healing has focused on decreased vascularity and hypoxia in affected tissues. More recently, impaired leukocyte function has been implicated as an additional factor in the pathophysiology of radiation injury. Reconstructive surgical plans may require alteration when operating in a radiated field, especially in the head and neck. Radiation wounds are best treated by standard, accepted techniques of thorough debridement and coverage with well-vascularized tissue. Hyperbaric oxygen may have a role in the management of early radiation injury or in prophylaxis against postoperative wound complications.

Induction of neutrophil Mac-1 integrin expression and superoxide production by the medicinal plant extract gossypol.

Gossypol is present in antiinflammatory poultices made from the medicinal tree Thespesia populnea. Isolated human neutrophils exposed to 3-20 microM gossypol for 15-90 min were assayed in vitro for superoxide production and surface expression of Mac-1 (CD11b/CD18). Gossypol increased superoxide production in a time- and concentration-dependent fashion consistent with a moderate, delayed respiratory burst. Surface Mac-1 expression was increased within 15 min by 3-5 microM gossypol, resulting in a 14-fold increase over controls and a threefold greater increase over that produced by PMA. Staurosporine failed to block gossypol induction of superoxide and Mac-1, while EDTA inhibited induction of Mac-1 only, implicating a calcium-dependent mechanism. Gossypol increased intracellular calcium to peak levels, but in a delayed fashion as compared to FMLP. These findings demonstrate that gossypol is a highly potent stimulant of Mac-1 expression and suggest at least two protein kinase C-independent pathways of neutrophil activation. The resultant exhaustion of neutrophils may account for the antiinflammatory properties of plants containing gossypol.

Chest wall reconstruction.

Chest wall defects are frequently encountered in all regions of the chest. Initial defect assessment includes evaluation of location, extent, and etiology of the defect. Reconstructive options include flap transposition, tissue expansion, and microvascular composite tissue transplantation. Partial thickness defects are readily covered with skin grafts if viable muscle is present in the wound base. Complex defects, particularly related to wound débridement for osteomyelitis or osteoradionecrosis, are covered with regional muscle or musculocutaneous flaps. Extensive full-thickness defects frequently require restoration of the bony defect. Split rib grafts are preferred for elective sterile extirpative defects. Complex defects with unfavorable wound environment (chronic open wound or osteoradionecrosis) may require use of Prolene mesh to maintain chest wall stability and to provide support for the overlying flap. Tissue expansion is useful for partial thickness defects in order to provide optimal contour and skin quality at the site of reconstruction and to avoid additional scars at distant donor sites (see Fig. 6). Microsurgical composite tissue transplantation will allow complex defect closure when regional muscle or musculocutaneous flaps are unavailable. With careful defect analysis in regard to reconstructive requirements, the surgeon may select appropriate options from the reconstructive triangle to accomplish safe chest wall reconstruction with optimal form and function.

Update on chronic osteomyelitis.

Recent advances in the understanding of the pathophysiology and treatment of osteomyelitis have been discussed. Some of the advances (in either knowledge or technology) that we have found particularly useful include the following: The discovery of additional prognostic factors, which allow more reliable preoperative assessment. The development of the Ilizarov device, which allows bone fixation and later limb lengthening. Confirmation of the effectiveness of single-staged debridement and muscle flap closure. Confirmation that a brief (10 to 14 day) course of antibiotics is as effective as more prolonged therapy. The development of several new free muscle donor sites and a greater appreciation of the need for careful insetting of the muscle. The development of local antibiotic delivery systems, especially continuous antibiotic irrigation catheters. The development of newer oral and intravenous antibiotics that allow outpatient therapy following surgery. The recognition that patients require extended follow-up, and that any recurrences can be successfully treated with a second debridement and muscle flap closure.

Advances in muscle and musculocutaneous flaps.

The use of the muscle and musculocutaneous unit as a reconstructive tool by the plastic and reconstructive surgeon is expanding to all body regions. The initial four basic factors required in use of the muscle flap for reconstructive surgery now include such modifications as: (1) transfer of the sensory nerve to the musculocutaneous unit with the flap for sensory preservation; (2) use of minor vascular pedicles as a point of rotation for certain muscle flaps, (3) use of combinations of more than one muscle flap with common proximal vascular supply; and (4) transfer of the bony origin of the muscle unit with the muscle flap. Refinements and further additions to this list are demonstrated throughout this text and will certainly increase as the muscle flap is adapted for use in coverage of difficult wounds. An accurate knowledge of the vascular anatomy of muscles will allow safe modifications in the design of the muscle and musculocutaneous unit by the reconstructive surgeon.