Nerve allograft regeneration and immunological tolerance of rats with sciatic nerve transplantation after intragastric pretreatment with ultraviolet B-irradiated donor splenocytes

BACKGROUND： Nerve allograft rejection is an unavoidable problem for nerve allografts. Traumatic peripheral nerve injuries are commonly reconstructed using autogenous nerve grafts. However, this form of reconstruction is limited by insufficient autologous nerves for large gap repairs and by morbidity at the nerve donor site. OBJECTIVE： To examine sciatic nerve regeneration and immune tolerance reaction after intragastric administration of ultraviolet B-irradiated （UVB） donor splenocytes. DESIGN, TIME AND SETTING： A complete randomized grouping design and controlled experiment. The experiments were conducted in the Department of Orthopedics, the First Affiliated Hospital to Shanxi Medical University, China, between March and October 2007. MATERIALS： Fourteen adult male SD rats and fourteen male Wistar rats, weighing 250-300 g, were randomly matched as donors and acceptors. A further seven male SD rats （weight 250-300 g, age 12-16 weeks） were used for nerve isografts. Immune preparations and the Epics XL flow cytometer were purchased from B-D Company, USA. A computer-assisted electromyograph machine was provided by Keypoint P, Dantel Company, Denmark. METHODS： Splenocytes from Wistar rats were isolated, purified, and cultured, and then irradiated with ultraviolet B. In the first control group （Group 1）, the SD rats received a syngeneic SD nerve isograft. In the second control group （Group 2）, the SD rats received a nerve allograft from Wistar rats without pretreatment. In the oral-tolerance treated group （Group 3）, the SD recipient rats were inoculated with 2.5×10^7 Lewis UVB-irradiated donor splenocyte cells by intestinal tract administration at seven days before transplantation. MAIN OUTCOME MEASURES： （1） The recent end and the middle and distal end of the transplanted nerve were cut at 8 and 12 weeks after operation. Recovery of nerve regeneration was measured with HE staining using the total number, density, and diameter of the nerve fibers. （2） The level of CD25＋T lymphocytes in peripheral blood was detected with the Epics XL flow cytometer at one week after operation. （3） The bilateral sciatic nerves were exposed from the sciatic notch up to 0.5 mm beyond the distal graft site at eight weeks post-engraftment. Bipolar platinum stimulating electrodes were placed under the sciatic nerve proximally and the mean conduction velocity was recorded with recording electrodes on the posterior tibial nerve at 0.3 cm distal to the nerve graft. RESULTS： Eight weeks after operation, total axon number and fiber density in Group 3 were higher than that in Group 1 （P 〈 0.05）, neural regeneration in Group 3 was lower than that in Group 1 （P 〈 0.05） , The level of CD25＋T lymphocytes in peripheral blood of Group 3 was significantly lower than that of Group 2 （P 〈 0.05）. There was no significant difference between Group 3 and Group 1 （P 〉 0.05）. At eight weeks post-engraftment the mean conduction velocity of Group 3 approximated that of Group 1. The untreated allografts in Group 2 demonstrated no measurable recovery of conduction velocity. CONCLUSION： Pretreatment with a single intragastric dose of UVB-modified donor antigen specifically induced tolerance to peripheral nerve allografts in rats.

Allogeneic peripheral blood stem cell transplantation in the treatment of severe aplastic anemia and severe infection

Objective To investigate the efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) in the treatment of severe aplastic anemia (SAA) and severe infection. Methods A patient with SAA and pseudomonas aeruginosa septicemia was treated with PBSCT from an HLA-identical sibling with cyclophosphamide (CY) and total body irradiation (TBI) for conditioning. The patient was infused with 20.3×108/kg mononuclear cells including 61.0×106/kg CD34+cells following the conditioning regimen. Results Twelve days after PBSCT, the absolute neutrophil count (ANC) of 1.0×109/L was achieved, with platelet count >50×109/L at twenty days. The donor origin of engraftment was confirmed by polymerase chain reaction (PCR) analysis of short tandem repeats at the end of the first, sixth and twelfth month. The patient’s body temperature dropped to normal level when her ANC reached 0.5×109/L on day 10, and the bacterial culture of blood sample became negative subsequently. Symptoms and signs of acute or chronic graft versus host disease (GVHD) were not observed in 30 months after PBSCT. Conclusions Hematopoiesis was reconstituted shortly after PBSCT. The combination of CY and TBI and the infusion of sufficient peripheral blood stem cells may contribute to the successful engraftment. PBSCT may be considered as the first choice when hematopoietic stem cell transplantation is needed for SAA patients complicated with severe infection.

A preliminary pathological study on human allotransplantation

Objective: To observe the survival of hand allograft under the state of immunosuppression and the pathological changes of rejection in the recovery process. Methods: The biopsies of the skin, nerve, muscle, tendon and bone tissue of hand allografts during different stages from 1 day to 7 months after operation were observed using routine histological technique. Results: No significant changes due to rejection in skin, nerve, muscle and bone tissue were observed. But different degrees of weak rejective changes were found on the wall of blood vessels; in the muscle and nerve the reactions were markedly stronger than those found in skin tissues. Conclusions: The rejection in deep tissues should be monitored in controlling the rejection of hand allograft.

The effect of FasL expression on pancreatic islet allografts

OBJECTIVE: To investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in pancreatic islet allografts. METHODS: Allogeneic islets and testicular cells were cotransplanted into diabetic recipients.Pancreatic islets were infected with the recombinant adenovirus, AdV-FasL, and transplanted into diabetic recipients. Allograft survival, islet function, apoptosis of infiltrative lymphocytes in allografts and gene transfected islet allografts were analyzed. RESULTS: All animals receiving islet allograft alone returned to a diabetic state in a few days (mean survival time 6.3 +/- 0.6 days). When the quantity of testicular cells cotransplanted with islets increased to 1 x 10(7), all animals remained normoglycemic throughout the follow-up period (60 days). FasL expression by cotransplanted Sertoli cells induced apoptosis of activated lymphocytes. Rejection of allografts in the FasL gene transfer group was accelerated and allograft survival was shortened to 3.4 +/- 0.2 days (P

Psychological consequences derived during process of human hand allograft

OBJECTIVE: To study the psychology and the treatment during the process of hand allograft. METHODS: The patients were interviewed to evaluate their states of mind and their abilities to manage stress during the selection of patients. The psychology of the two patients were trained before the operation and managed accordingly afterwards. RESULTS: One of 12 candidates was found to be unsuitable for the transplantation because of psychiatric problems. One week postoperatively, the two patients were anxious, lacked patience and were afraid of seeing the long-awaited grafted hand. After 1 week, the patients began to accept the new hand, with full acceptance of the hand 1 month later. With the recovery of hand sensation 4 to 5 months after the operation, the patients regarded the hand as their own. CONCLUSION: Psychological problems exist during preoperative selection of patients and postoperative rehabilitation, requiring psychologists in the hand transplantation team.

Vertebral plate regeneration induced by radiation-sterilized allogeneic bone sheets in sheep

Objective： To evaluate the effects and mechanism of radiation-sterilized allogeneic bone sheets in inducing vertebral plate regeneration after laminectomy in sheep. Methods ： Twelve adult male sheep （ aged 1.5 years and weighing 27 kg on average ） provided by China Institute for Radiation Protection underwent L3-4 and L4-5 laminectomy. Then they were randomly divided into two groups： Group A （n =6） and Group B （n =6）. The operated sites of L4-5 in Group A and L3-4 in Group B were covered by ＂ H-shaped＂ freeze-drying and radiationsterilized allogeneic bone sheets （ the experimental segments）, while the operated sites of L3-4 in Group A and L4-5 in Group B were uncovered as the self controls （ the control segments ）. The regeneration process of the vertebral plate and the adhesion degree of the dura were observed at 4,8,12,16,20 and 24 weeks after operation. X- ray and CT scan were performed in both segments of 1.34 and L4-5 at 4 and 24 weeks after operation. Results： In the experimental segments, the bone sheets were located in the anatomical site of vertebral plate, and no lumbar spinal stenosis or compression of the dura was observed. The bone sheets were absorbed gradually and fused well with the regenerated vertebral plate. While in the control segments, the regeneration of vertebral plate was not completed yet, the scar was inserted into the spinal canal, compressing the dura and the spinal cord, and the epidural area almost disappeared. Compared with the control segments, the dura adhesion degree in the experimental regenerated segments was much milder （ P 〈 0. 01 ）, the internal volume of the vertebral canal had no obvious change and the shape of the dura sack remained well without obvious compression. Conclusions： Freeze-drying and radiation-sterlilized allogeneic bone sheets are ideal materials for extradural laminoplasty due to their good biocompatibility, biomechanical characteristics and osteogenic ability. They can effectively reduce formation of post-laminectomy scars, prevent recurrence of post-lamlnectomy spinal stenosis, and induce regeneration of vertebral plates.

A biomechanical investigation on the incorporation of cortical allograft in rabbit ulna defects

To explore the changes of biomechanical properties of cortical allograft in different mechanical environments. Methods: Cortical allograft was transplanted to each side of the midshaft diaphyseal ulna of each one of 40 rabbits. The left transplanted allograft underwent normal physiological load, while the right one underwent lower load. After animals were killed,specimens were taken for examination of bone mineral density, bone porosity and maximal three-point-bend breaking load. Results: The union strength of allograft-host bone junction was increased constantly; meanwhile, the internal creeping substitution led to an initial greater weakening of the cortical allograft itself and a later recovery of its strength. In comparison, the union strength of the normally loaded graft-host bone construct was significantly higher than that of the lower loaded side at the 8th and 16th week after transplantation. At the 16th week, there was greater bone strength in normally loaded graft than that in lower loaded graft. Conclusions: The internal repair can lead to initial greater weakening of cortical allograft and later gradual recovery of its strength. The effect of physiological load can accelerate the improvement of the biomechanical properties of allograft.