Rashid A. Fawwaz

Induction of donor-specific unresponsiveness to rat cardiac allografts by intrathymic injection of UV-B-irradiated donor spleen cells

This study examined the role of intrathymic injection of allogeneic spleen cells in induction of donor-specific unresponsiveness to heart allografts in the Lewis-to-ACI rat combination. Intrathymic injection of naive Lewis SC led to rejection in naive or sublethally irradiated (200 rads TBI) ACI recipients at times equivalent to those obtained in control animals. Intrathymic injection of UV-B-irradiated Lewis SC, on the other hand, led to indefinite cardiac allograft survival (>300 days) in sublethally irradiated ACI recipients; similar treatment failed to prevent rejection of third-party (Wistar Furth) cardiac allografts, which demonstrates the specificity of the immunologic unresponsiveness thus induced. The finding that intrathymic injection of untreated allogeneic SC does not prevent rejection of subsequently transplanted allograft suggests that modulation of major histocompatibility complex class II molecule by methods such as UVB may be critical to induction of unresponsiveness. Inoculation of UV-B donor SC in extrathymic sites (subcutaneous, intraperitoneal and intratesticular) did not significantly prolong graft survival in similarly prepared animals, thus confirming the privileged position of the thymus in the induction of tolerance. When the unresponsive recipients of cardiac allografts were made diabetic at 100 days and rechallenged with a second-set donor-type neovascularized pancreatic islet grafts, three of four animals accepted permanently (>100 days) the islet grafts, thus indicating tolerance to donor alloantigens. To define the underlying mechanisms of specific tolerance in this model, in vitro MLR and in vivo adoptive transfer studies failed to demonstrate suppressor activity in the long-term cardiac allograft recipients. In contrast CML assays using 51Cr-release showed that T cells obtained from the unresponsive animals had no detectable cytotoxic activity to Con A-stimulated donor blast targets. The latter finding suggests clonal anergy or deletion of cytotoxic T cells to donor alloantigens. Our results confirm the role of the thymus as a privileged site for the induction and maintenance of specific immunologic unresponsiveness to organ allografts and suggest that this approach may be potentially useful in clinical transplantation of immediately vascularized allografts and neovascularized grafts.

Induction of donor-specific unresponsiveness to rat cardiac allografts by pretreatment with intrathymic donor MHC class I antigens

Since intrathymic injection of UV-B-irradiated spleen cells induces donor-specific unresponsiveness in the sub-lethally irradiated (200 rads TBI) recipients, while intrathymic injection of naive SC leads to acute graft rejection, we hypothesized that presentation of MHC class I rather than MHC class II antigens to immature T cells in the thymus may convey a tolerogenic signal to the recipient. The present study was designed to examine if intrathymic injection of naive MHC class I—positive resting T lymphocytes can induce antigen-specific unresponsiveness to cardiac allografts in the Lewis-to-ACI rat combination. The results showed that intrathymic injection of resting Lewis T cells consistently induced indefinite graft survival (>300 days) in sublethally irradiated (200 rads TBI) ACI recipients while similar treatment failed to prevent the rejection of third-party (Wister-Furth) cardiac allografts, thus demonstrating the specificity of the immunologic unresponsiveness to donor alloantigens. Examination of the timing of intrathymic antigen presentation relative to cardiac transplantation that would achieve 100% permanent graft survival in the Lewis-to-ACI rat combination showed that the optimal time was 7 days before allografting, while peritransplant and immediate post-transplant intrathymic inoculation of donor T cells was relatively ineffective in the induction of unresponsive-ness to donor grafts. We also showed that removal of the antigen-containing thymus in the sublethally irradiated recipients with functioning cardiac allografts consistently caused graft rejection if performed earlier than 21 days after heart transplantation; thymectomy after 21 days of organ transplantation did not affect indefinite survival of the grafts. Thus, it appears that the maintenance of peripheral tolerance to the grafts after 21 days of transplantation may be dependent on the presence of a new clone of antigen-specific tolerant host T cells. These results confirm the immunologic privileged position of the thymus in the induction of central and peripheral tolerance, and suggest that pretreatment with intrathymic MHC class I alloantigens is potentially useful in the induction of unresponsiveness to donor vascularized allografts in adult animals and in man.

A Phase I Study of 90Yttrium-Ibritumomab-Tiuxetan in Children and Adolescents with Relapsed/Refractory CD20-Positive Non–Hodgkin's Lymphoma: A Children's Oncology Group Study

Purpose: The prognosis for children with recurrent CD20+ non–Hodgkins lymphoma is dismal. A radiolabeled anti-CD20 antibody, 90yttrium-ibritumomab-tiuxetan (90Y-IT), is Food and Drug Administration approved for adults with recurrent indolent CD20+ B cell–non–Hodgkins lymphoma. There is no data on the safety and feasibility of 90Y-IT in refractory childhood CD20+ lymphoma. Experimental Design: Children and adolescents with refractory/relapsed CD20+ lymphoma were eligible for this phase I radioimmunotherapy study. Patients (n = 5) received rituximab (250 mg/m2 i.v.) on days 0 and 7 and indium-111 ibritumomab-tiuxetan (5 mCi i.v.) on day 0. Dosimetry studies were done on days 0, 1, 3, and 6. Immediately after rituximab on day 7, patients received 90Y-IT if dosimetry studies showed <2000 cGy exposure to all solid organs and <300 cGy to marrow, as well as 0.4 mCi/kg in patients with good marrow reserve (n = 3) and 0.1 mCi/kg in patients with poor marrow reserve (after bone marrow transplant; n = 2). Results: No patients experienced nonhematologic or hematologic dose-limiting toxicity. Human antimurine antibody/human antichimeric antibody incidence was 0%. One patient experienced grade II infusion–related chills associated with rituximab. The following are the means of organ radiation exposure (cGy): kidneys 341 (112-515), liver 345 (83-798), lungs 309 (155-519), marrow 46 (20-78), spleen 565 (161-816), and total body 42 (14-68). Conclusions: Based on these findings, an expanded investigator-initiated limited institutional phase II study has been designed to further evaluate the safety, tolerability, and response rate with 90Y-IT dose stratification based on marrow reserve.

Use of indium-111-labeled cells in measurement of cellular dynamics of experimental cardiac allograft rejection

This study evaluates the kinetics and utility of infused indium-111-labeled cells in detecting rejection in ACI to Lewis rat heart allografts. Syngeneic leukocytes, lymph node lymphocytes, and platelets were isolated and labeled with indium-111 (111In) oxine, respectively, and were infused i.v. into Lewis rats carrying beating ACI or syngeneic hearts from post-transplant days 0 to 6. Recipients were imaged serially at 24 hr after infusion of labeled cells followed by excision of both native and transplanted hearts for direct isotope count. Labeled leukocytes accumulate progressively in the allograft with the scan becoming positive by post-transplant day 4. The ratio of allograft to native heart isotope counts rose from 1.25 on day 1 to 10.07 (P < 0.0001) on day 7. The Lewis recipients infused with labeled lymphocytes showed a positive scan on days 6 and 7 whereas the allograft to native heart isotope count ratio rose from 0.97 on day 1 to 5.33 (P < 0.001) on day 7. Recipients infused with 111In-labeled platelets showed a positive scan on days 5 to 7 and the allograft to native heart isotope count ratio rose sharply from 2.56 on day 4 to 16.98 (P < 0.005) on day 7. Syngeneic heart grafts failed to demonstrate significant accumulation of any of the labeled cell population. These studies confirm the importance of nonlymphocytic cells in cellular rejection, evaluate the kinetics of graft invasion by the various cell types, and suggest that the techniques used afford a method for a safe and an early detection of allograft rejection.

Selective lymphoid irradiation: III. Prolongation of cardiac xenografts and allografts in presensitized rats

Selective lymphoid irradiation (SLI) with palladium-109-hematoporphyrin (Pd-H) combined with antilymphocyte globulin (ALG) induces either donor-specific permanent rat heart allograft acceptance or significant allograft prolongation depending on the degree of donor-recipient matching. The purpose of this study was to determine if SLI combined with ALG can affect ACI heart allograft survival in Lewis recipients presensitized to ACI, and of hamster heart xenografts of Lewis rats. SLI combined with ALG delays allograft and xenograft rejection in the presence of induced or preformed antidonor antibodies, and converts primarily a humoral rejection into a cellular rejection by mechanisms as yet uncertain. Such peritransplant treatment had significant effect on the levels of antidonor complement-dependent cytotoxic antibody titers but did not correlate directly with graft survival. Histological analysis of rejected hearts in all groups demonstrated primarily a humoral hyperacute rejection in control animals and in recipients treated with ALG alone, while peritransplant treatment with Pd-H and ALG resulted not only in prolonged graft survival but histologically, primarily a cellular rejection of the graft. (JMT)

Migration patterns of dendritic cells in the rat: Comparison of the effects of γ and UV-B irradiation on the migration of dendritic cells and lymphocytes☆

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.

SINGLE PHOTON EMISSION CT AND POSITRON EMISSION TOMOGRAPHY IN THE EVALUATION OF NEUROLOGIC DISEASE

Widely available SPECT allows imaging of certain critical components of neurotransmission, providing clinically and experimentally significant information. Future efforts may be directed toward developing innovative techniques to delineate dynamic neurochemical changes in vivo.

Mn (III) hematoporphyrin. A potential MR contrast agent.

Manganese (III) hematoporphyrin (MnHP), a new and stable complex, was prepared, and its toxicity and magnetic resonance (MR) imaging properties were evaluated. In tests of acute and subacute toxicity, no deaths resulted from bolus intravenous injections of 13 or 19 mumols/kg of MnHP, but there was a 33% mortality when the dose was 38 mumols/kg. Laboratory results were normal in the surviving rats. Ultraviolet- visible spectroscopy of the urine and serum of two rats injected 24 hours previously with 38 mumols/kg MnHP revealed no free HP, suggesting in vivo stability of MnHP. Finally, using a standardized imaging protocol, there was a mean increase of 37% in the liver-to-muscle intensity ratios in four rats injected 24 hours previously with 25 mumols/kg MnHP when compared to paired controls (P less than .005). In addition, obvious visual increase in the signal intensity of the liver on T1-weighted images was seen in animals tested with 13 and 19 mumols/kg of MnHP. The results suggest that further evaluation of MnHP as an MR contrast agent for the liver is warranted.

The use of radionuclides for tumor therapy

The successful use of radionuclides for tumor therapy depends to a major extent on the ability to achieve a high concentration of radioactivity in the tumor relative to other radiosensitive organs not involved by tumor, such as bone marrow, intestinal mucosa, liver, and kidneys. Techniques designed to achieve such differential localization of the radionuclides include the use of radiopharmaceuticals that enter specific metabolic pathways unique to certain tumor types; radiolabeled antibodies that attach to tumor-associated antigens present on tumor cell surfaces; heterologous antibodies that attach to tumor-associated antigens present on tumor cell surfaces and which are then identified by radiolabeled antibodies directed against the species in which the original, unlabeled antibody was made, and radiolabeled compounds injected regionally at the tumor site. Although both clinical and experimental evidence on the use of radionuclides for tumor therapy is encouraging in preliminary studies, extensive further research needs to be done in this area to insure the clinical efficacy of radionuclides for tumor therapy.

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine.

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 108 donor leukocytes (DL) combined with peritrans-plant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5–8 per group, were pretreated with 108 DL with or without peritransplant CsA. A single DL transfusion on day −3 or day −7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0±0.9 days in controls to 12.2±4.5 days and 12.4±1.0 days (P< 0.01), respectively. Two DL infusions on days −7 and −3 or on days −14 and −7 prolonged the MST to 10.6±1.3 days (P<0.02) and 16.4± 2.8 days (P<0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1±2.7 days. When pretransplant DL transfusion on day −3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (>100 days). Similarly, DL infusion on day −7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days −7 and −3 combined with CsA resulted in indefinite graft survival (>100 days) in 4/6 animals. Transfusion of DL on day −3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days −14 and −7 led to significant donor graft survival of 9.8±0.4 days (P<0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3±0.8 and 10.0±1.1 days, respectively. Passive transfer of 1108 spleen cells obtained at 20 and 100 days after transplantation from recipients of long-term cardiac allografts prolonged the donor test grafts in naive hosts to 13.5±2.6 and 10.8±1.9 days, respectively. Transfer of both sera and spleen cells obtained at 20 days after transplantation had no effect on a third-party (ACI) heart allograft survival. These results suggest that pretransplant DL infusion combined with a brief peritransplant CsA can induce immunologic unresponsiveness that is dependent on collaboration between serum-specific suppressor factor or antibody, and the generation of donor-specific suppressor cells. This study reemphasizes the importance of leukocytes in immunologic manipulation using donor-specific transfusions and describes the strong synergistic effect of CsA on the use of DL.