Maciej Zielinski

Donor-specific tolerance in fully major histocompatibility complex-mismatched limb allograft transplants under an anti-αβ T-cell receptor monoclonal antibody and cyclosporine A protocol

Background. Recent studies have demonstrated that treatment with &agr;&bgr;–T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined &agr;&bgr;-TCR–CsA protocol. Methods. The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of &agr;&bgr;-TCR and CsA or a combination of &agr;&bgr;-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. Results. Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined &agr;&bgr;-TCR–CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (A×C Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. Conclusions. Combined therapy of &agr;&bgr;-TCR–CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.

Induction of tolerance to hind limb allografts in rats receiving cyclosporine A and antilymphocyte serum: effect of duration of the treatment.

Background. This study assessed the ability of antilymphocyte serum (ALS) and cyclosporine A (CsA) to induce tolerance for hind limb composite tissue allograft in rats without chronic immunosuppression. Methods. Hind limb transplantations were performed in Lewis-Brown-Norway (LBN, RT1l+n) and Lewis (LEW, RT1l) rats. Treatment consisted of ALS only (0.4 mL/kg), CsA only (16 mg/kg), and a combination of CsA and ALS, and it was administered 12 hr before surgery at three different intervals (7, 14, and 21 days). Long-term survivors were tested for tolerance by standard skin grafting from the recipient (LEW), the donor (LBN), and the third party (ACI, RT1a) 60 days after cessation of the treatment and by mixed lymphocyte reaction at 100 days. T-cell lines were analyzed with flow cytometry. Results. Single use of ALS in all treatment intervals did not prolong allograft survival. Single use of CsA extended survival up to 23 days in the 21-day protocol group. CsA and ALS caused indefinite survival in two of six rats in the 14-day protocol and in all six rats in the 21-day protocol (>420 days). The six long-term survivors in the 21-day protocol accepted the skin grafts from the donor (LBN) and the recipient (LEW) and rejected third-party grafts (ACI). Tolerant animals showed a donor-specific hematopoietic chimerism of 35% to 42% in the peripheral blood. Mixed lymphocyte reaction assay demonstrated tolerance to the host and donor alloantigens and increased response to the third party. Conclusions. Administration of CsA and ALS for 21 days induced donor-specific tolerance in the recipients of the rat hind limb composite tissue allografts. The mechanism of tolerance should be investigated further.

Comparison of clinical evaluation and neurosensory testing in the early diagnosis of superimposed entrapment neuropathy in diabetic patients

Diabetic patients are more susceptible to the development of entrapment neuropathy than nondiabetics. Since these patients suffer from a slowly progressing diabetic polyneuropathy, standard neurosensory and motor tests of nerve function are not sufficient in the diagnosis of superimposed nerve compression. This is most evident in the early stages of compression when quantitative diagnosis is important for making decisions on surgical decompression. We evaluated the validity of computer-assisted pressure-specified sensory device (PSSD) testing in the early detection of superimposed entrapment in diabetic neuropathy in comparison with standard clinical tests. Twenty-five diabetic patients with complaints of peripheral nerve dysfunction were evaluated by clinical tests and PSSD. Out of those, nerve entrapment was detected in 15 patients (60%) (9 in late and 6 in early stage) by neurosensory PSSD testing. Standard clinical tests were confirmative in 33.3% of these cases (44% of late and 16.7% of early stage). Out of 144 evaluated nerves, 50 were diagnosed with entrapment (24 in late and 26 in early stage) using PSSD. Clinically, diagnosis was confirmed in 16% of entrapped nerves (20.8% of late and 11.5% of early stage). Average diabetes duration in patients with entrapment diagnosed using PSSD was significantly shorter than for those diagnosed clinically (4.14 ± 2.04 vs. 7.2 ± 1.3, respectively; P = 0.005). Among evaluated factors, mean age and diabetes duration were found to be significantly shorter in patients with entrapment than in those with advanced diffused changes (54.47 ± 13.07 vs. 67.10 ± 14.2; P = 0.019 and 5.33 ± 3.74 vs.14.22 ± 8.17; P = 0.006; respectively). Our results revealed higher sensitivity of PSSD in comparison with standard clinical tests in the detection of early-stage entrapment in patients with diabetes. To assess accuracy of PSSD in the proper patients’ qualification for surgery, further prospective, postoperative studies are needed.

A novel technique for peripheral nerve repair

Objective To evaluate a novel technique for the repair of neural deficits using a single fascicle to bridge an injury in the rat sciatic nerve.

The single-fascicle method of nerve grafting.

In this study a single-fascicle technique for neural deficits repair was evaluated using a rat sciatic nerve model. Twenty-four Lewis rats were divided into 4 groups: group 1, 1.5-cm deficit without repair; group 2, conventional autograft; group 3, large-fascicle autograft; and group 4, small-fascicle autograft. Nerve regeneration was evaluated by pin-prick and toe-spread tests. Nerve samples were estimated by histomorphometry. Group 1 presented no recovery. Groups 3 and 4 demonstrated significantly better pin-prick results compared with those from conventional repair. Histology revealed a significantly higher number of axons and myelin thickness in the small-fascicle (2.8 ± 0.4 × 103 axons, 4.22 ± 0.41 &mgr;m) and large-fascicle (5.1 ± 1.7 × 103 axons, 4.62 ± 0.28 &mgr;m) groups compared with the conventional autograft group (2.1 ± 0.3 × 103 axons, 2.93 ± 0.20 &mgr;m). The small-fascicle group had a significantly greater mean axon area (58.59 ± 15.81 &mgr;m2) than the large-fascicle group (29.66 ± 12.67 &mgr;m2) and the conventional group (25.35 ± 7.52 &mgr;m2). In this study, peripheral nerve repair using a single-fascicle graft resulted in faster functional recovery and better morphometric outcome compared with conventional nerve repair.

Development of Mouse Cremaster Transplantation Model for Intravital Microscopic Evaluation

Objective: In this study, we investigated the possibility of transplanting cremaster muscle in mice. After transplantation, we measured the microcirculatory parameters and compared with the cremaster‐control values with no transplantation.

216. Development of donor specific chimerism and tolerance in composite tissue allografts under ab-t cell receptor monoclonal antibody and cyclosporine a treatment protocols

Purpose Recently, we induced donor specific tolerance to rat hind-limb allografts under 35 days course of ab TCR mAb and Cyclosporine A. In this report, we investigated the role of shorter ab-TCR/CsA protocols on the tolerance induction. Materials and Methods We performed fifty-two hind-limb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21, 7 and 5 day protocols of combined ab-TCR/CsA treatment on tolerance induction. Donor specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor specific chimerism was assessed by flow cytometry. Results All transplants under 5,7, and 21 days of combined ab-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytornetry revealed high level of donor chimerism in the peripheral blood of the long term survivors. Conclusion The extention of survival of limb allografts and allounresponsiveness were directly associated with the development of stable chimerism in the tolerated recipients.

214. Induction of the donor specific chimerism and tolerance in fully MHC mismatched limb allograft recipients

Purpose Induction of donor-specific tolerance in composite tissue allografts (CTA) remains one of the most challenging goals in transplant surgery. Recently we have developed a protocol (ab TCR mAb/ CsA) for tolerance induction in the semiallogeneic rat hind-limb transplantations!. In this report we induced tolerance in the fully MHC mismatched allograft recipients under, combined ab-TCR/CSA protocol. Methods Thirty transplantations across strong MHC barrier were performed between Brown-Norway donors (RT1n) and LEW recipients. Isograft and allograft rejection control groups received no treatment. Experimental groups received either rat abTCR or CsA or combination of abTCR and CsA at the day of transplantation and for 7 days thereafter. The efficacy of immunosuppressive treatment and chimerism were monitored by flow cytometry FC. Donor specific tolerance and immunocompetence of the limb recipients were determined in vivo by secondary skin graft from the recipient (LEW), the donor (BN) and the third party (ACI) graft. Mixed lymphocyte reaction (MLR) was performed for the assessment of donor specific tolerance in vitro. Results Only fully mismatched allograft recipients under abTCR/CsA protocol survived indefinite (over 250 days). Three color FC analysis at day 120 post-transplant demonstrated stable, multilineage, donor specific chimerism in the periphery of the tolerated recipients (CD4+PE/RT1n+APC-7.6% and 1.3% of CD8+PE/RT1n+APC positive T cell sub-populations and CD45RA+PE/RT1n+APC-16.5% B cell population). Donor specific tolerance and immunocompetence in vivo was confirmed by acceptance of the secondary skin graft from the donor and rejection of the third-party grafts. MLR revealed no reactivity to host (LEW) and donor (BN) antigens but strong reactivity to the third-party alloantigens. Conclusions Donor specific tolerance was induced under 7 days protocol of abTCR/CsA therapy in the fully mismatched limb allograft transplanrs. Stable mixed chimerism was achieved without the need for the myeloablative bone marrow modification of the recipients and without the need for chronic immunosuppression.