Effect of mutants and inhibitors on mitochondrial transport systems in vivo in yeast.

We have reported elsewhere (Wills, C. and Martin, T. (1984) Biochim. Biophys. Acta 782, 274-284) that one or more mitochondrial transport systems may be involved in the regulation of the inducible alcohol dehydrogenase of yeast, ADH-II. In order to investigate this phenomenon further, it was necessary to determine which of these systems operate in the cell in vivo. We give in this paper preliminary evidence that inhibitors of the malate-phosphate (n-butyl malonate), malate-citrate (hydroxycitrate) and malate-alpha-ketoglutarate (aminooxyacetate or cycloserine) transport systems all operate in vivo. While the demonstration of the in vivo inhibitory activity of n-butyl malonate and hydroxycitrate is entirely by physiological methods, that of the transaminase inhibitors aminooxyacetate and cycloserine depends in part on the isolation of mutants capable of growth on glycerol in minimal medium. On this medium these mutants depend on the malate-aspartate shuttle for growth, and as expected the transaminase inhibitors prevent their growth. Two of the mutants show an enhanced rate of mitochondrial glutamate uptake. A preliminary survey of the properties of the glycerol growth mutants is presented, showing that the probable mode of action of these mutants is an increase in the efficiency of the malate-aspartate shuttle.

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.

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.

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.

Multilineage cells from human adipose tissue: implications for cell-based therapies.

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

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.

Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads.

Multipotential processed lipoaspirate (PLA) cells extracted from five human infrapatellar fat pads and embedded into fibrin glue nodules, were induced into the chondrogenic phenotype using chondrogenic media. The remaining cells were placed in osteogenic media and were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (BMP-2). We evaluated the tissue-engineered cartilage and bone using in vitro techniques and by placing cells into the hind legs of five severe combined immunodeficient mice. After six weeks, radiological and histological analysis indicated that the PLA cells induced into the chondrogenic phenotype had the histological appearance of hyaline cartilage. Cells transfected with the BMP-2 gene media produced abundant bone, which was beginning to establish a marrow cavity. Tissue-engineered cartilage and bone from infrapatellar fat pads may prove to be useful for the treatment of osteochondral defects.

Stimulus preprocessing and response selection in depression: a reaction time study.

Depressed subjects are slower than normal controls in reaction time (RT) tasks. However, it is not clear whether depression affects all stages of information-processing or only some of them. In the present study, this question was addressed by using the additive factor method. Ten inpatients and ten control subjects performed a two-choice visual RT task. Stimulus intensity and stimulus-response compatibility were manipulated. The effect of intensity was similar in both groups whereas the effect of compatibility was larger for the patients than for the controls. This suggests that stimulus preprocessing is unaffected by depression whilst response selection is impaired.

Comparison of long-term immunosuppression for limb transplantation using cyclosporine, tacrolimus, and mycophenolate mofetil: implications for clinical composite tissue transplantation.

This study compared the efficacy of long-term intermittent immunosuppression in preventing the rejection of a limb transplant across the strongest histocompatibility barrier in ACI --> Lewis rats using the conventional immunosuppressive agent cyclosporine-A and the newer immunosuppressive agents FK-506 (tacrolimus) and RS-61443 (mycophenolate mofetil). The recipient animals were immunosuppressed daily for 14 days postoperatively, followed by long-term intermittent, twice-weekly immunosuppression using cyclosporine 25 mg/kg, RS-61443 30 mg/kg, or FK-506 2 mg/kg. All three immunosuppressive agents were able to prolong the rejection of the skin component of a limb transplant compared with nonimmunosuppressed controls. Eight of nine animals receiving cyclosporine immunosuppression showed signs of rejection of the skin component of the limb transplant while continuing to receive long-term immunosuppression and had a mean rejection time of 61.6 days. Seven of 10 animals immunosuppressed with RS-61443 also showed signs of rejection while still receiving immunosuppression, with a mean rejection time of 43.6 days. Nine of 10 animals receiving FK-506 immunosuppression showed no signs of skin rejection, but died of bacterial pneumonia between 273 and 334 days after transplantation, with a mean rejection time of 296.1 days. There was no statistically significant difference between intermittent immunosuppression with cyclosporine and RS-61443, but FK-506 was significantly superior to both cyclosporine and RS-61443. The implication of this study is that FK-506, but not cyclosporine or RS-61443, is probably the only single immunosuppressive agent capable of preventing rejection of the skin component of a composite tissue transplant. Combination immunosuppression with FK-506 and RS-61443, therefore, may be required to allow composite tissue transplantation to become a predictable clinical reality in the future.

An experimental model to determine the effect of irradiated tissue on neutrophil function.

Complications of irradiated tissue include infections and impaired healing. Although fibrosis and hypovascularity contribute, a cellular mechanism has not been identified. This study examines the effect of radiation (10 to 30 Gy) on neutrophil function in a rabbit wound cylinder model. At 3 to 12 weeks after radiation, subcutaneous wound cylinders were implanted in both irradiated and control fields in 19 rabbits. Wound neutrophils were subsequently assayed for phagocytosis (3H-labeled Staphylococcus aureus assay), superoxide production (cytochrome c reduction assay), and surface Mac-1 expression (flow cytometric assay using MHM 23 monoclonal antibody). Phagocytosis of 3H-labeled S. aureus was significantly lower in neutrophils from irradiated fields compared with controls at 6 and 12 weeks after radiation (6.5 versus 18.9 bacteria per neutrophil at 12 weeks; p = 0.027). Stimulated neutrophils from irradiated tissue could not increase superoxide production or Mac-1 expression as much as controls, with differences increasing as postirradiation time increased. The diminished phagocytosis, superoxide production, and Mac-1 expression provide a cellular mechanism that may account for susceptibility to infection and poor healing in irradiated tissues.

Donor leg morbidity and function after fibula free flap mandible reconstruction.

The purpose of this study was to determine the donor leg morbidity and function after removal of the fibula free flap for mandible reconstruction. In the past 24 months, 29 consecutive patients underwent a total of 30 fibula free flap mandible reconstructions. A muscle-sparing technique was used to harvest the fibula flap, and the proximal 6 cm and distal 8 cm of fibula were left intact. Patients included 20 men and 9 women; their mean age was 58.8 years (range 29 to 82 years); the mean length of fibula removed was 14.5 cm (range 8 to 25 cm); osteocutaneous flaps were used in 27 patients (90 percent); and 16 patients (53 percent) required skin grafts to the donor leg. Donor leg morbidity and function were determined by patient questionnaire, physical examination, and isokinetic testing, with the opposite, unoperated leg serving as a control. Immediate postoperative morbidity occurred in 5 patients (17 percent) (infection, wound separation, or partial graft loss); none required additional surgery for donor complications. Patient questionnaires were completed by all patients at an average of 7.3 months after surgery. Patients were able to ambulate pain-free an average of 5.1 weeks (range 2 to 32 weeks) postoperatively and were all fully able to engage in all daily and recreational activities. Most (21 patients, 72 percent) were free of any donor pain, and the remainder (28 percent) had only occasional mild discomfort. Other complaints included ankle stiffness (41 percent), mild ankle instability (10 percent), and transient peroneal motor (7 percent) or sensory (28 percent) loss, which resolved in all patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural resistance to infection: leukocyte functions.

The nonspecific immune system has an extraordinarily important role in prevention of and response to wound infection. In the presence of injury or contamination, leukocytes adhere to local endothelial cells in response to complement signals through an integrin mechanism; they then migrate to the affected tissue site to locate, ingest, and kill microbes. Clinically, the major weaknesses in this system are impairment of cell recruitment and depression of microbial killing mechanisms; intracellular killing is the most problematic step in the process. Provided that leukocytes are able to migrate and ingest bacteria, nonoxidative killing proceeds in a normal fashion. Oxidative killing, however, can be profoundly impaired by poor blood perfusion and oxygenation. Low oxygen tension is a common clinical problem in surgical sites and wounds. There are a number of ways in which oxidative killing can be supported and facilitated in the clinical arena in a manner that is likely to enhance the effects of antibiotics in the treatment and prevention of infection.

[Control of the rejection process treated with cyclosporine and RS-61443, in allogeneic microsurgery transplantation of a limb, in rats]. / Controle do processo de rejeição em transplante alógeno microcirúrgico de membro tratado com ciclosporina e RS-61443, em ratos.

Composite tissue allotransplantations would contribute to the reconstructions of the congenital, traumatic and tumors deformities. PURPOSE--The aim of this study is to prevent or inhibit the rejection in limb vascularized allotransplantation; decrease the toxicity of available immunosuppressive agents; determine whether combination subtherapeutic doses of CsA +RS-61443 will have an additive immunosuppressive effect. METHODS--Five groups were studied, using Brown-Norway limb donors and Fischer 344 recipient rats: group A Untreated autograft controls; group B Untreated allograft controls; group C Allografts: CsA 1.5 mg/kg/d SQ; group D Allograft: RS-61443 15 mg/kg/d; group E Allograft: combination CsA + RS-61443. RESULTS--The results were the following concerning the rejection: Group A animals displayed no rejection clinically (0%). All the skin biopsies obtained were devoid of rejection (grade 0). Group B animal developed rejection at 10-13 days post-transplantation. Skin biopsies confirmed the epidermal necrosis (grade 4). Group C animals developed rejection in 55%. Group D animals developed rejection in 94%. In contrast, Group E animals had 96% rejection-free survival up to POD 172 thus far. CONCLUSION--Combination subtherapeutic doses of CsA + RS-61443 was effective in preventing acute rejection of limb allografts and had an additive immunosuppressive effect because of the agents immunosuppressive synergistic effect.

Vibration absorbing brace for study of work-related upper extremity musculoskeletal disorders.

The use of hand-held power tools can result in absorption of significant vibration energy by the worker's hand and arm, and is a causal factor in the development of various muskuloskeletal disorders (MSD's) such as carpal tunnel syndrome (CTS) and hand-arm vibration syndrome (HAYS). A novel brace incorporating vibration damping materials is proposed that could reduce this energy absorption, resulting in lower incidence of vibration-related occupational MSD's. The National Occupational Research Agenda (NORA) has identified this type of injury as a priority research area. The proposed brace will have utility in the top three NORA-sponsored areas for research tools and approaches: clinical assessment of exposure to environmental vibration; practical use as improved personal protective gear; and evaluating the effects of reduced vibration exposure on MSD incidence. Clinical test results for prototype braces on human volunteers are reported: splints with piezoelectric material showed a median improvement of 12% in vibration damping as compared to otherwise identical nonpiezoelectric splints (median allowable error of +/- 1.5%). The use of these splints to further study how the hand-arm system absorbs vibration energy is also discussed.

Posttraumatic chylous ascites in a child: recognition and management of an unusual condition.

Chylous ascites is an extremely rare and often unrecognized complication of abdominal trauma in children. The management of this condition has traditionally been nonsurgical, but the success rate with nonoperative treatment is not always satisfactory. A case of posttraumatic chylous ascites in an abused toddler is presented, with emphasis on the diagnosis and treatment of this rare disorder.

Down-regulation of decorin, a transforming growth factor-beta modulator, is associated with scarless fetal wound healing.

PURPOSE: Transforming growth factor beta (TGF-beta) bioactivity has been implicated as a potential regulator of the transition from scarless healing to scar formation in fetal wounds. Decorin is an extracellular matrix proteoglycan that regulates TGF-beta bioactivity and assists in collagen fibrillogenesis. To determine its role in scarless repair, the authors examined decorin expression in fetal fibroblasts, skin, and wounds. METHODS: A single, full-thickness, 2-mm open wound was created on the dorsal surface of fetal rats at 16.5 days (E16) and 18.5 days (E18) gestational age (term, 21.5 days [E21]). Wounds were harvested at 24 and 72 hours (n = 12 wounds per time-point). Nonwounded fetal skin at E17, E19, and E21 was harvested for analysis of decorin expression during skin development and as controls for wounds. In addition, fetal (E14, E18) and adult dermal fibroblasts were cultured for in vitro analysis. Reduced-cycle, specific primer, reverse transcriptase polymerase chain reaction was performed to quantitate decorin expression. RESULTS: Decorin expression increased rapidly with increasing gestational age in both fetal fibroblasts and skin. Expression was increased 22-fold in E18 fibroblasts (P <.002) and 300-fold in adult fibroblasts (P <.001) compared with E14 fibroblasts. In skin, expression increased 74% (P <.01) during the fetal wound healing transition period between E17 and E19. However, in E16 wounds (scarless), decorin expression decreased 59% (P <.006) at 24 hours and 45% (P <.02) at 72 hours. Decorin expression did not change in E18 (scar) wounds at 24 and 72 hours (P >.05). CONCLUSIONS: Early gestation fetal fibroblasts and fetal skin express decorin at lower levels than late gestation fetal and adult fibroblasts and skin. Decorin expression is down-regulated in scarless (E16) compared with scar (E18) wounds. Thus, increased decorin expression is associated with both skin development and scar formation. Conversely, decreased decorin expression is associated with scarless repair.

Controle do processo de rejeiçäo em transplante alógeno microcirúrgico de membro tratado com ciclosporina e RS-61443, em ratos / Control of the rejection process treated with cyclosporine and RS-61443, in allogeneic microsurgery transplantation of a limb, in rats

Os transportes alógenos de tecido composto contribuiram sobremaneira para reparaçäo das deformidades congênitas, traumáticas e tumorais. OBJETIVO. Prevenir ou inibir o processo de rejeiçäo em transplante microcirúrgico alógeno de membro; diminuir a toxicidade das drogas imunossupressoras; verificar um melhor efeito imunossupressor através da possibilidade do efeito sinérgico da associaçäo de doses subterapêuticas de ciclosporina e RS-61443. MÉTODOS. Cinco grupos foram estudados, usando Brown-Norway, doadores e Fischer 244, receptores: grupo A, controle autógeno sem tratamento; grupo B, alógeno, sem tratamento; grupo C, alógeno + ciclosporina 1,5mg/Kg/d sc; grupo D; alógeno + RS-61443 15mg/Kg/d; e grupo E, alógeno + CsA + RS. RESULTADOS. De acordo com o processo de rejeiçäo, os resultados foram o seguinte: grupo A, nenhum animal apresentou rejeiçäo (0 por cento). Todas as biópsias resultaram em grau 0; grupo B, todos desenvolveram rejeiçäo entre o 10º e o 13º dia pós-operatório. As biópsias cutâneas confirmaram a necrose epidérmicas (grau 4); grupo C, 55 por cento desenvolveram rejeiçäo; grupo D, 94 por cento apresentaram rejeiçäo e em contraste; no grupo E, 94 por cento dos animais sobreviveram livres do processo de rejeiçäo, por mais de 172 dias de transplante. CONCLUSäO. A associaçäo de duas drogas imunossupressoras (ciclosporina + rs-61443), em doses subterapêuticas, foi eficiente em prevenir o processo de rejeiçäo e apresentou um melhor efeito imunossupressor pelo efeito sinérgico da associaçäo destas drogas

The incidence of lethal graft-versus-host disease in rat limb allotransplantations

To study graft-versus-host disease (GVHD) in rat hindlimb allotransplantation, a model similar to intestinal transplantation was used. ACI and Lewis rats were crossed to produce F(1) generation rats (ACI-Lewis). These underwent limb transplantation receiving a donor limb from a Lewis rat. The animals were examined daily and skin biopsies were performed. Results showed that all limbs were viable at 34 weeks and no rats demonstrated any clinical evidence of GVHD and biopsies were grade 0.

Rat limb allotransplantation: efficacy of subtherapeutic dose combination immunotherapy with cyclosporine (CsA) and RS-61443

The synergic effect of subtherapeutic doses of cyclosporine and RS-61443 was demonstrated in a vascularized rat hindlimb allotransplantation across a strong histocompatibility barrier (Brown-Norway as donors and Fischer 344 as recipients). Low doses of agents in combination minimized the toxicity while increasing the therapeutic efficacy. All animals showed weight loss during the first 15 days posttransplantation and they regained protective sensation within 45 postoperative days. Only 15.38 per cent of the animals presented complications: thrombosis, enteritis, autophagia and disorders of unknown etiology.