Leg Transplantation Research Articles

The Effect of Dose-Fractionation Mode on the Immune Response and Therapeutic Effect of Lewis Lung Cancer Mice Model Based on Tumor Microenvironment

The therapeutic effect of different groups of Lewis lung cancer mice and the expression of immune cells in peripheral blood and tumor tissues of mice were observed after different dose-segmentation modes of treatment, so as to select the most suitable for stimulating immune effect with good prognosis and small side effects. Female homologous inbred C57BL/6 male mice at 6-8 weeks were selected and inoculated subcutaneously on the right thigh according to LLC: L929: lymphocyte = tumor cell suspension with PBMC(inactivated lymphocyte) = 5:1:1 ratio; after tumor growth to the size of 100-200mm3, different dose-segmentation modes were used for irradiation, which was divided into 5 groups, group A: no radiotherapy; Group B: 2Gy/qd, continuous radiotherapy for 30 days; Group C: 2Gy/bid, continuous radiotherapy for 13 days; Group D: 3Gy/bid, continuous radiotherapy for 10 days; Group E: 6Gy/qd, continuous radiotherapy for 8 days. On the 3rd day after radiotherapy, some mice were sacrificed, peripheral blood lymphocytes of mice were isolated with lymphocyte separation solution, and the transplanted tumor of mice was prepared into single-cell suspension. Then, the number of CD3+T cells, CD3+B cells, NK1.1 cells, CD3+CD4+T cells, CD3+CD8+T cells, CD4+Foxp3+Treg cells in peripheral blood and single-cell suspension of mice was detected by flow cytometry. HE staining was used to observe the tumor tissue structure of mice leg transplantation. The expressions of CD4, CD8, Foxp3 and CD31 in tumor tissues of each radiotherapy group were observed by immunohistochemical staining (IHC) in paraffin sections. The survival time of mice in group C (2Gy/ bidx13d) was significantly improved compared with that in group B (2Gy/qdx30d), and the twin-daily segmentation mode could improve the elevation of Treg cells after once-daily segmentation mode radiotherapy in both peripheral blood and tumor tissues. There was no significant difference in the survival time of mice in group E (6Gy/qdx8d) and group D (3Gy/bidx10d), the number of Treg cells in peripheral blood of mice in group E was significantly lower than that in group D, but there were more Treg cells in tumor tissue of mice in group E than group D. Compared with the control mice, the overall immunity of the experimental group was inhibited. The twice-daily segmentation mode in the conventional dose radiotherapy group significantly improved Treg in peripheral blood and tumor immune microenvironment and promoted neovascularization. The twice-daily segmentation mode in the high-dose radiotherapy group improved the immunosuppression of lymphocytes in peripheral blood but increased immunosuppression in tumor microenvironment.

Mesenchymal Stem Cell Therapy to Promote Sensory and Motor Nerve Function in a Limb Transplant Model.

Introduction: Hand and leg transplantation (Tx) in military/civilian populations is increasingly needed to improve the quality of life of individuals. Optimizing nerve regeneration and preventing immune rejection are vital to successful outcomes in limb Tx. As a first step, our objective was to investigate whether bone marrow derived mesenchymal stem cells (MSCs) can improve nerve regeneration and function. Methods: We performed orthotopic syngeneic hind-limb transplants in Lewis (RT1. Al) rats. Osteosynthesis of femur was performed using intramedullary pin and bone cement. The donor and recipient vessels were anastomosed using the vascular cuff technique. The sciatic nerve ends were approximated using 10-O nylon sutures. Rats received syngeneic MSC (5x106; passage ≤8; n=8) or vehicle (n=7) locally (nerve, bone and vascular anastomoses sites) and intravenously. Following Tx ( ≥2 weeks) laser doppler and radiologic analysis were performed to assess limb vascularization and fusion of native and graft femoral ends. Sensory function of the transplanted limb was assessed by cutaneous pain reaction test and motor function by walking track analysis every 1-2 weeks until 26 weeks post-Tx. Results:Ex vivo expanded Rat MSCs were CD29+, CD31-, CD34-, CD44+, CD45low, CD90+, MHC Class I+, Class II- and were pluripotent: differentiated in to adipocytes, osteocytes and chondrocytes in ex vivo cultures. Limb Tx was highly successful (100% survival). Four weeks post-Tx sensory nerve function was <0.3 on a scale of Grade 0 to 3 (0=No function; 3=Normal function). By 8 weeks in vehicle group it was 2.2±0.7 (tibial), 1.2±0.5 (peroneal) and 1.7±0.9 (sural), and in MSC group it was 2.6±0.4 (tibial), 1.0±0.9 (peroneal) and 1.7±0.9 (sural). Overall sensory function was higher but not significant in MSC group compared to vehicle group at 4, 8, 16 and 24 weeks post-Tx. Walking track analysis (for motor nerve function) did not produce clear foot prints to calculate Sciatic Function Index (SFI), so a novel grading system for available foot prints was developed. Radiology and laser doppler analysis revealed normal bone fusion and vascularization, respectively. Gastrocnemius muscle was atrophied (P<0.05) and animals developed flexion-contractures in the transplanted limb. Conclusion: The limb transplantation procedure was highly successful, sensory function was recovered (>70%) and MSC therapy appears to promote transplanted limb nerve functional recovery.

Stem cell therapy to promote sensory and motor nerve function recovery in a limb transplant model (TRAN3P.863)

Abstract Introduction: Hand and leg transplantation (Tx) in military/civilian populations is increasingly needed to improve the quality of life. Our objective was to investigate whether Mesenchymal Stem Cell (MSC) therapy can improve nerve regeneration and function. Methods: We performed orthotopic syngeneic hind-limb transplants in Lewis (RT1.Al) rats. Following Tx rats received syngeneic MSC (5x106; passage ≤8) or vehicle intravenously and locally. Results: Ex vivo expanded Rat MSCs were CD29+, CD31-, CD34-, CD44+, CD45low, CD90+, MHC Class I+, Class II- and pluripotent. Limb Tx was highly successful (100% survival). Four weeks post-Tx sensory nerve function (determined by cutaneous pain reaction) was &amp;lt;0.3 on a scale of Grade 0 to 3 (0=No function; 3=Normal function). By 8 weeks in vehicle group it was 2.2±0.7(tibial), 1.2±0.5(peroneal) and 1.7±0.9 (sural), and in MSC group it was 2.6±0.4(tibial),1.0±0.9(peroneal) and 1.7±0.9(sural). Overall sensory function was higher but not significant in MSC group compared to vehicle group at 4, 8, 16 and 24 weeks post-Tx. Walking track analysis (for motor nerve function) did not produce clear foot prints to calculate Sciatic Function Index (SFI), so a novel grading system was developed. Gastrocnemius muscle was atrophied (P&amp;lt;0.05) and animals developed flexion-contractures in the transplanted limb. Conclusion: The limb Tx procedure was highly successful, sensory function was recovered (&amp;gt;70%) and MSCs appear to promote nerve function recovery.

19-P010 Head-regeneration through cell death and compensatory proliferation in the Hydra

cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. In contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. Since these findings fit well with the Ds/Ft steepness model for the determination of organ size, we propose the following model for the control of leg regeneration. A Ds/Ft gradient is responsible for sensing positional disparity and driving proliferation of regenerating cells by activation of the Hippo/Warts pathway. The leg size of regenerates is determined by the steepness of the Ds/Ft gradient, such that growth ceases when the slope of the linear gradient falls below a certain threshold value, which may be determined by an Ex/Mer interaction. Our findings provide additional insight into the longstanding question of how the size and shape of an appendage is determined in metazoa.

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg.

Resistance to acute rejection by shRNA interference from CD40 costimulatory molecule in limb allo-transplantation in rats

Objective To study the resistance to acute reaction in composite tissue allotransplan-tation by shRNA interference from CD40 costimulatory molecule in rat lymphocytes. Methods The in-bred SD rats were chosen as donors and inbred Wistar rats as recipients. Twenty-seven recipients were divided into 3 groups randomly after allogeneic leg transplantation. On the day after transplantation, rats in group A were injected with 600 μL of Sofast-siCD40-2/pSilencer, rats in group B received 600 μL of Sofast-pSilencer 4.1-CMV neo, and rats in group C received injection of 600 μL of sodium chloride. The survival and rejection to grafts were observed. The rats which survived 7 days after operation were given the test of mixed lymphocyte reaction (MIR). Results The mean survival time of limb allografts in group A was obviously longer than that in the other two groups( P ＜ 0.01 ) . Rejection to grafts was not observed in group A, but rejection e-merged shortly after transplantation in the other two groups. Rats in group A had a low reaction to the donors' lymphocytes. Conclusion Without any immnosuppressants, shRNA interference from CD40 costimula-tory molecule can resist acute rejection in limb allotransplantation in rats. Key words: Tissue; Transplantation; RNA interference; Graft survival; Graft rejection

Résultat à long terme d’une replantation de jambe

Leg replantation is generally considered to yield good results, particularly since the widespread use of progressive lengthening procedures. This optimism must be weighted against the very long and difficult operative program, the social and occupational implications, and the functional outcome of the replanted limb. We report a case of below knee leg replantation in a young patient whose operative program lasted 29 months. Occupational activities were interrupted for three years. The clinical result was satisfactory but with 14̊ residual valgus suggesting a potential risk of osteoarthritis and difficult therapeutic decisions. The alternatives to replantation are controversial. Leg transplantation is not a valid option in our opinion. Amputation with orthesis should be considered more often because complications are rare and recovery much shorter. Indications for leg replantation should be considered with prudence.

From Leg Transplantation by St Cosmas and St Damian to the Modern Era

From Leg Transplantation by St Cosmas and St Damian to the Modern Era

Leg transplantation in rats

Leg transplantation in rats