Samuel Yu

Induction of donor-specific tolerance to rat nerve allografts with portal venous donor alloantigen and anti-ICAM-1/LFA-1 monoclonal antibodies

BACKGROUND Expendable autologous nerve available for repair of nerve injuries is limited. Cadaveric allografts could provide adequate nerve for grafting but are susceptible to rejection and currently require chronic systemic immunosuppression. METHODS Age-matched Buffalo (RT1B) rats randomly assigned to 7 experimental groups received either a Lewis (RT1L) rat posterior tibial-sciatic nerve allograft or a Buffalo (RT1B) syngeneic isograft. Recipients were treated with anti-intercellular adhesion molecule-1 (anti-ICAM-1) and anti-leukocyte function-associated antigen-1 (anti-LFA-1) monoclonal antibodies (mAbs), intraportally administered ultraviolet B (UVB)-irradiated donor splenocytes, a combination of mAbs and UVB-irradiated donor splenocytes, or no immunosuppression. RESULTS Histologic, electrophysiologic, and functional evaluation of nerve allografts 8 weeks after transplantation revealed a synergistic improvement in nerve allograft regeneration when monoclonal antibodies were combined with UVB-irradiated donor splenocytes. In vitro immunologic assessment correlated with the in vivo allograft function by demonstrating a donor-specific suppression of the recipients response in mixed lymphocyte culture (MLC), cytotoxic T lymphocyte assay (CTL), and reduced T cell interleukin-2 production in both the group receiving UVB-irradiated alloantigen alone and the group receiving a combination of UVB antigen and mAbs. CONCLUSIONS The combined use of mAbs directed against ICAM-1 and LFA-1 adhesion molecules with intraportally administered UVB modified donor antigen prevents nerve allograft rejection and synergistically improves nerve regeneration.

Brief cyclosporine treatment prevents intrathymic (IT) tolerance induction and precipitates acute rejection in an IT rat cardiac allograft model.

BACKGROUND Intrathymic (IT) alloantigen combined with administration of rabbit anti-rat anti-lymphocyte serum (ALS) intraperitoneally induces donor-specific tolerance to rat cardiac transplants. The purpose of this study was to examine the effect of a brief course (4 days) of cyclosporine (CsA) on the development of IT tolerance. METHODS Buffalo (BUF) (RT1b) rats were given 25x10(6) fully MHC-mismatched Lewis (LEW) (RT1l) splenocytes by IT injection plus 1.0 ml of ALS intraperitoneally. Twenty-one days later, IT donor-specific LEW (group 1) or third-party (ACI, RT1a) (group 2) hearts were heterotopically transplanted to the abdominal aorta A third group of BUF (group 3) were given daily CsA (10 mg/kg) by oral gavage for 4 days before administration of IT LEW cells and ALS. Rejection as defined by the cessation of a palpable heartbeat was confirmed by histology. Cytokine profiles of allografts from all groups were then analyzed using a multi-probe RNase protection assay. RESULTS Sixty-seven percent of IT/ALS-treated BUF recipients not pretreated with CsA accepted LEW heart grafts for greater than 90 days. However, 86% of animals treated with CsA for 4 days before IT injection and ALS rejected allografts at 10.7+/-3.2 days. Third-party allografts (ACI) were uniformly rejected (7.0+/-0.0 days). Histology confirmed cellular rejection in CsA-treated allografts and cytokine analysis detected increased interleukin (IL)-3, IL-5, and tumor necrosis factor-alpha when compared to increased IL-2 and interferon-gamma in rejecting untreated controls. CONCLUSIONS CsA can prevent the induction of intrathymic alloantigen tolerance. These results support the development of a CsA-sensitive, but IL-2-independent, active regulatory mechanism after intrathymic exposure to donor-specific alloantigen and depletion of mature peripheral T cells.

The Kinetics Of Tolerance Induction By Nondepleting Anti-cd4 Monoclonal Antibody (rib 5/2) Plus Intravenous Donor Alloantigen Administration 1,2

BACKGROUND CD4+ T cells play an essential role in allograft rejection. The monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating the recipient T cells. We have successfully induced tolerance to rat heart allografts by recipient pretreatment with a single dose of RIB 5/2 plus intravenous administration of donor splenocytes. In this study, we explored whether this potent regimen could induce tolerance to the more resistant kidney and skin allografts. Furthermore, we examined the kinetics and requirements for tolerance to be met by a single dose of RIB 5/2 plus i.v. alloantigen. METHODS The efficacy of a single i.p. dose of 20 mg/kg RIB 5/2 plus i.v. donor antigen (25x10(6) splenocyte) pretreatment 0, 21, or 40 days before receipt of an MHC-mismatched Lewis (RT1l) to Buffalo (RT1b) rat cardiac, renal, or skin allograft was studied. Another group of Buffalo recipients treated with RIB 5/2 plus an i.v. alloantigen +/-thymectomy received kidney transplants after 40 days. Attempts to prevent tolerance used interleukin-2 or prior sensitization. Mixed lymphocyte cultures, cytotoxic assays, and precursor frequencies of helper and cytotoxic cells, by limiting dilution analysis, serially measured in vitro cell-mediated immunity. RESULTS RIB 5/2 administration combined with i.v. alloantigen 21 days before induced tolerance to heart and kidney allografts but did not prolong skin graft survival. In contrast, kidney allografts delayed for 40 days after pretreatment were acutely rejected and survival was not affected by the thymectomy. MLC, CTL, and pTH, and pCTL precursor frequencies from recipients of long-term grafts were specifically suppressed to donor, but not third party, alloantigen. CONCLUSION A single dose of the nondepleting anti-CD4 monoclonal antibody, RIB 5/2, plus i.v. alloantigen is a potent inducer of tolerance to heart and kidney, but not skin, allografts. The RIB 5/2-induced donor unresponsiveness to a delayed kidney or cardiac allograft is time dependent but can be prolonged if specific alloantigen is present. Suppression of cell-mediated allo-immune responsiveness correlates with allograft acceptance.

Pretreatment With Portal Venous Ultraviolet B Irradiated Donor Alloantigen Promotes Donor‐Specific Tolerance to Rat Nerve Allografts

Objective To determine if a single intraportal inoculation of ultraviolet B‐irradiated (UVB) donor splenocytes can prevent nerve allograft rejection and confer donor‐specific immunotolerance to rat nerve allograft segments.

Augmentation of cell-mediated cytotoxicity following 50% partial hepatectomy.

The cytolytic responses of C3H/HeJ mice after 50% hepatectomy (PH) were assessed in a 4-hr 51Cr-release assay. Spleen cells (SC) (50 x 10(6] from normal or PH C3H/HeJ (H-2k) mice were sensitized with equal numbers of irradiated allogeneic DBA/2 (H-2d) spleen cells in a five-day mixed lymphocyte culture. Generated cytolytic activity was measured against 51Cr-labeled P815 mastocytoma (H-2d) and EL4 lymphoma (H-2b) target cells. The wet weight and cell numbers per spleen following 50% partial hepatectomy were 70% and 75% higher than the control values for the first 20 days, and then returned to normal levels by 21 days. The cytolysis by spleen cells from 2-, 14-, and 31-day PH mice were 89.3 +/- 0.7, 86.9 +/- 5.3, and 90.1 +/- 1.3%, respectively, compared with control (sham-operated) values of 56.0 +/- 1.0, 57.0 +/- 2.0, and 49.9 +/- 7.0% (P less than 0.03 at E/T 100:1). This enhanced cytolysis by PH spleen cells remained high for at least 118 days after the liver resection before returning to control levels by 268 days. Cytolytic effector cells in PH SC were generated at least 24 hr earlier than in control SC. When normal and PH cytolysis were compared following primary and secondary in vitro sensitization, the cytolytic levels of primarily-sensitized PH spleen cells were comparable to secondarily sensitized normal spleen cells. Furthermore, the primarily sensitized normal spleen cells did not show crossreactive cytolysis with EL4 target cells (H-2b), while both the primarily sensitized PH spleen cells and the secondarily sensitized normal spleen cells were significantly cross-reactive against the third party EL4 target cells. Adherent PH spleen cells appear to be responsible for this augmented cytolytic capacity since their coculture with normal nonadherent responder spleen cells increased control cytolysis by approximately 30%. These studied demonstrate that, following 50% partial hepatectomy, there is an immediate and sustained increase in the allospecific cytolytic response.

Portal venous ultraviolet B–irradiated donor alloantigen prevents rejection in circumferential rat tracheal allografts☆

BACKGROUND: Before tracheal transplantation can be considered as a method of reconstruction in patients with extensive circumferential tracheal defects, we must achieve a state of nontoxic, donor-specific tolerance so that the risks of such a transplant do not outweigh the benefits. OBJECTIVE: Our objective was to determine whether a single intraportal injection of modified donor alloantigen achieves donor-specific immunosup-pression for major histocompatibility complex-mismatched rat tracheal allografts. STUDY DESIGN: Buffalo (recipient) rats were pretreated with either a single portal-vein administration of ultraviolet B (UVB)-irradiated donor splenocytes (n = 4) or an intraportal inoculation of nonirradiated donor splenocytes (n = 4). Major histocompatibility complex-mismatched Lewis (donor) tracheal allograft segments were then grafted into treatment groups 7 days after donor-cell pretreatment. Tracheal rejection was assessed by histologic analysis, mucosal cilia motility, and in vitro immunologic assessment. RESULTS: The UVB-treated group demonstrated no acute or chronic rejection as well as complete functional recovery. In vitro immunologic assessment demonstrated a donor-specific hyporesponsiveness and donor allospecificity. Untreated animals and those receiving nonirradiated donor splenocytes showed acute rejection of their tracheal allografts. CONCLUSION: Recipient pretreatment with intraportally administered UVB-irradiated donor splenocytes prevents rejection of circumferential rat tracheal allograft segments by inducing a donor-specific immune hyporesponsiveness.

Reversal of ischemic liver failure by intrasplenic liver cell transplantation.

A model of ischemic hepatic failure in Sprague-Dawley rats (SD) with 100% mortality has been developed by one-hour occlusion of the right portal vein and hepatic artery followed by left 70% hepatectomy. The intrasplenic injection of 40 x 10(6) syngeneic adult or three-day neonatal single liver cell suspensions decreased the mortality from 100% to 50% and 36%, respectively. Mortality decreased with increasing time from the intrasplenic injection of neonatal liver cells to the time of acute hepatic ischemia. Mortality also decreased with increasing interval between hepatic ischemia and removal of the transplanted liver cells by splenectomy. Intrasplenic injection of graded doses of neonatal liver cells decreased mortality from 75% at a dose of 10 x 10(6) cells, to 36% at 160 x 10(6) cells. Treatment of neonatal liver cells with metabolic inhibitors did not significantly affect their ability to reverse acute hepatic ischemia.

Restoration of lymphocyte proliferation and CTL generation by murine rIL-2 after treatment of allogeneic stimulator cells by ultraviolet B irradiation, heat, or paraformaldehyde

Following a 5-day mixed lymphocyte culture (MLC), C3H/HeJ (H-2k) splenocytes stimulated with DBA/2 (H-2d) gamma-irradiated splenocytes (2000 rads) are specifically cytotoxic in a 4-hr 51Cr-release assay to P815 (H-2d) target cells (62±2% cytolysis) but not to third-party EL4 (H-2b). However, when the DBA/2 stimulator cells were treated with heat inactivation (45°C for 1 hr), fixed with 1% paraformaldehyde (15 min), or irradiated with ultraviolet-B light (104 J/M2), no cell proliferation or cytolytic activity developed in the MLCs. The levels of IL-1, IL-2, and IL-6 from the supernatants of MLC using stimulators undergoing either of the three treatments were markedly decreased compared with that from 7-irradiated stimulators. Both cell proliferation and specific cytolysis were restored in a dose-dependent fashion by the addition of murine rIL-2 to the MLCs. If the stimulator cells were first activated with 5 tig/ml pokeweed mitogen or lipopolysaccharide for 2 days, the subsequent treatment with heat, paraformaldehyde, or UV-B did not significantly affect the development of cytolysis (54–70% cytolysis). Suppressor cells were not detected when cells from the nonresponsive MLCs (2.5×108 cells) were added to an MLC freshly prepared with γ-irradiated stimulator cells, or were injected intraperitoneally (50×106 cells) into naive mice 2 days before recovery and in vitro sensitization of splenocytes. Therefore, modification of the stimulating alloantigen can prevent the release of cytokines that function as an essential second signal in the development of the proliferative response and subsequent cytolysis. The cytokine found to be essential for restoration of this response is IL-2.

Inhibition of Cardiac Allograft Rejection by Continuous Local Infusion of 16,16 Di-Methyl PGE2

Previous studies have implicated prostaglandins as pivotal modulators of the immune response with special emphasis on their function as local feedback inhibitors of T lymphocyte activation in vivo and in vitro.1 Many of the earlier studies showed that prostaglandin E2 (PGE2) interactions with T cells in vitro resulted in elevations of the cyclic adenosine monophosphate (cAMP) level,2 and that such an elevated intracellular cAMP level in T cells mediated the proliferative disturbances.3–5