Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.

The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

Late adjunctive therapy with single doses of rapamycin in skin-allografted mice treated with antilymphocyte serum and donor bone marrow cells.

Temporary donor-specific unresponsiveness induced by treatment of skin allografted mice with antilymphocyte serum (ALS) and donor bone marrow cells (BMC) can be converted to long-term graft survival and tolerance by adding adjunctive immunosuppressive agents, including rapamycin (Rapa). Rapa was given in a suboptimal dosing schedule to probe several aspects of its use in the promotion of tolerization. Single doses were given at 2 weeks post-transplantation to recipients prepared with ALS and donor BMC. Graft survival was markedly prolonged in a dose-dependent fashion by day +14, Rapa doses ranging from 0.75 to 6.0 mg/kg. Indefinite (> 300 day) graft survival was observed in 26% of recipients given the highest Rapa dose. Short-term treatment with cyclosporin A (CsA) or deoxyspergualin (DSG) was ineffective when injected at this time. Rapa augmented the tolerizing effect of grafting with skin from newborn mice but had no significant additive or synergistic effects with the short course of CsA or with DSG given on days +1 to +3, even though the latter prolonged graft survival when added to the ALS/BMC protocol. Single doses of Rapa on day +1 also prolonged graft survival, but without any of the grafts surviving indefinitely. Later dosing on day +28 resulted in > 70% of grafts surviving > 300 days. Challenge grafting of these mice after day +300 resulted in delayed rejection of donor strain, but not third-party skin grafts. Rapa was very effective when given as widely spaced doses on days +14 and +49. Also, grafts showing the earliest signs of rejection could be rescued with a single Rapa dose in recipients treated with ALS and BMC but not ALS alone. Transfer of prolonged graft survival with spleen cells from ALS plus BMC treated recipients was not adversely affected by Rapa given to the suppressor-like spleen cell donors approximately 1 week before cell harvest. We conclude that the use of Rapa as an adjunctive agent in allograft recipients treated with ALS plus donor BMC is very flexible in terms of timing of administration, and that the drug can be effectively given as widely spaced doses or as a rescue agent after ALS/BMC treatment. Additionally, an active immunoregulatory mechanism induced by ALS/BMC treatment appears to be spared by Rapa.

Effect of rapamycin on renal allograft survival in canine recipients treated with antilymphocyte serum, donor bone marrow, and cyclosporine.

Rapamycin (Rapa) monotherapy can promote renal allograft survival in dogs, but it is very toxic. To attempt to augment the effectiveness of Rapa and reduce its toxicity in a tolerance induction protocol, canine renal allograft recipients were treated briefly with antilymphocyte serum (ALS), donor bone marrow cells (BMC), and a limited course of cyclosporine (CsA). Rapa had little effect when CsA-treated recipients were given ALS on days -5 to -1 and BMC on day +1. When combined with CsA given days +13 to +42, ALS on days -5 to +7, and BMC on day +10, Rapa at 0.3 mg/kg on day +8 plus alternate days +15 to +39 significantly increased overall survival and was compatible with long-term survival after immunosuppression (6 grafts, 1 graft > 212 days, 1 graft > 470 days). Rapa appeared to prevent early rejections that can occur during treatment with these ALS/BMC/CsA protocols. Little toxicity of Rapa was observed with any treatment.

The effect of antilymphocyte serum, fractionated donor bone marrow, and cyclosporine on renal allograft survival in mongrel dogs.

The effect of combined treatment with antilymphocyte serum, fractionated donor bone marrow, and a limited course of cyclosporine on renal allograft survival in mongrel dogs was examined. Recipients were treated with ALS from day -5 to day +7, relative to transplantation on day 0 with an MLR-mismatched donor. Fractionated donor bone marrow cells (BMFr3) obtained by density gradient separation were infused 3-7 days after ALS treatment. CsA treatments were begun either 3-7 days after ALS plus BMFr3 infusion or 3-7 days after treatments with ALS alone. Extended allograft survival was achieved at all CsA doses in BMFr3-infused, ALS-treated recipients. Allografts were sustained throughout the CsA treatment period without rejection in the majority of recipients (6 of 8) receiving ALS plus BMFr3 and CsA at 20 Mg/kg/day for 60 days. By contrast, few grafts were sustained through 30 days of treatment with CsA at 3.2 (1 of 12) or 10 mg/kg/day (2 of 9) in ALS plus BMFr3-treated recipients. Cyclosporine treatment was ineffective at all doses in augmenting allograft prolongation in ALS-treated recipients that did not receive BMFr3. Nearly all (6 of 7) long-term survivors (greater than 180 days) were BMFr3-treated. Peripheral blood lymphocytes or bone marrow cells of these long-term survivors proliferated normally in response to Con A and PWM, and were MLR responsive to third-party cells but did not have reduced MLR responsiveness to donor alloantigen in all cases. These long-term survivors promptly rejected third-party renal allografts without adverse effects on the original transplant's function. These results show that long-term renal allograft survival with specific unresponsiveness to the donor can result from the combined treatment with ALS plus donor BMFr3 and a limited course of CsA.

Effect of posttransplantation administration of peripheral blood lymphocytes in skin-grafted mice treated with antilymphocyte serum or antilymphocyte serum plus bone marrow.

Posttransplantation administration of donor-specific bone marrow cells to ALS-treated allograft recipients produces graft survival that is greater than that produced by ALS treatment alone. We have now studied the effect of peripheral blood lymphocytes (PBLs) in a mouse skin allograft model using this protocol (C3H skin grafted to B6AF1 mice, day 0; i.p. ALS on days -1 and +2; i.v. bone marrow days +6 or +7). When PBLs are injected in place of bone marrow cells, graft survival is extended beyond that noted for control mice given ALS only. The timing and specificity of this phenomenon suggest that it resembles the effect produced by posttransplant bone marrow administration and not that associated with pretransplant blood transfusion. The PBLs active in graft prolongation are Thy-1-negative and display a density in Percoll gradients similar to that of the active marrow cells. In contrast, when PBLs are injected in combination with bone marrow cells, the length of graft survival is shortened in comparison with that produced by bone marrow alone. The cells associated with this partial abrogation of the effectiveness of bone marrow appear to be mature T cells; this abrogation cannot be produced by PBLs treated with anti-Thy-1 plus complement or by thymocytes, but it is a property of lymph node cells enriched for T cells by nylon-wool fractionation. This study suggests that clinical application of this posttransplantation induction of specific allograft unresponsiveness can be facilitated by the use of peripheral blood lymphocytes rather than marrow, sparing a living organ donor from having to undergo bone marrow harvest. Additionally, the data indicate that contamination of marrow with blood lymphocytes should be minimized. However, the density gradient fractionation method that we have found to be effective in preparing the graft prolonging bone marrow cells simultaneously removes the PBLs deleterious to graft survival.

Prolongation of graft survival in ALS-treated mice by donor-specific bone marrow. Density gradient fractionation of the active bone marrow cells.

Antilymphocyte serum (ALS)-treated recipient mice that are infused with donor bone marrow cells have been shown to enjoy prolonged skin graft survival. In the present experiments, discontinuous density gradient centrifugation in Percoll was used to prepare fractions of these cells. Two of four fractions had good graft-prolonging ability. In experiments in which the density of the gradients was varied, we were able to estimate the density of a major portion of the cells active in graft prolongation as being in a range of 1.061 to 1.066 g/ml, and to produce one fraction with superior graft-prolonging activity. This fraction constituted only 10% of the recovered cells, was enriched for small-to-medium lymphocytes, Ia+, Thy-1+, and IgM+ cells, and contained cells bearing a marker known to be present on active cells, Fc gamma R. Additionally, this fraction contained cells capable of suppressing the mixed lymphocyte reaction, although two denser fractions, of poor-to-good graft-prolonging ability, also shared this ability. The effectiveness of this method in bringing about a high-capacity, rapid isolation of cells that can augment graft survival in immunosuppressed recipients makes it well suited for future clinical application.

Specific tolerance to canine renal allografts following treatment with fractionated bone marrow and antilymphocyte serum.

These results suggest (1) that ALS + BM fractions can be used to promote tolerance in long-term graft survivors; (2) that the presence of apparent BM cells of donor origin in the allograft in normally functioning kidneys may be related to the mechanism of allograft prolongation, and (3) that some promise of possible specific long-term immunosuppression exists clinically.

Prolongation of renal allograft survival in antilymphocyte-serum-treated dogs by postoperative injection of density-gradient-fractionated donor bone marrow.

Posttransplant injection of fractionated donor bone marrow cells results in the prolonged survival of mouse skin allografts. In this investigation, we extended the study to the use of donor bone marrow fractionated by density-gradient centrifugation in a larger animal model, canine renal transplantation. Renal allografts were placed in outbred, histoincompatible dogs treated daily with rabbit or horse antilymphocyte serum (ALS) from days -6 to +7 relative to kidney transplantation on day 0. Fresh, unfractionated donor bone marrow (BM) or a bone marrow fraction (BM Fr3) produced by centrifugation in a discontinuous Percoll density gradient was intravenously infused into recipients on days +13 or +14. Alternatively, frozen/thawed (F/Th) BM or BM Fr3 was infused after storage at -80 degrees C for two weeks. BM Fr3 and unfractionated BM significantly (P less than 0.005) prolonged for median allograft function time (MFT) beyond the controls treated only with ALS (46 and 35 days vs. 18 days). The longer MFT with BM Fr3 was achieved with 20-40% of the unfractionated BM cell dose. In addition, BM Fr3-treated dogs exhibited a slower rate of loss of kidney function. F/Th BM was as effective as fresh BM in prolonging graft survival. A reduced mixed lymphocyte response to donor and third-party cells and markedly reduced responsiveness to Con A, PHA, and pokeweed mitogens at 30-45 days posttransplant suggested that animals were nonspecifically immunosuppressed during this period. By 60 days posttransplant, in two dogs treated with BM Fr3, the MLR to third-party cells, but not to donor cells, and the responsiveness to mitogens had returned to pre-ALS treatment values. Thus, treatment with ALS combined with fresh or F/Th BM that has been fractionated is an effective method for the prolongation of kidney allografts in dogs. The development of specific, long-term immunosuppression induced by fractionated bone marrow and success with F/Th BM in this canine renal allograft model suggests that application to human transplantation may be effective with fresh or F/Th BM fractions obtained from living-related or cadaveric donors.

Mouse skin graft prolongation with donor-strain bone marrow and antilymphocyte serum. Effect of bone marrow cell storage.

Significant extended survival of C3H/He skin grafts in antilymphocyte serum (ALS)-treated B6AF1 mice can be brought about by the injection of donor-strain bone marrow on day 6 or 7 after grafting. In the present study, survival of the active graft-prolonging bone marrow cells under several storage conditions was investigated. The bone marrow cells retained their effectiveness if stored at 4 degrees C in 10% fetal calf serum for 18 hr prior to injection, but not if maintained at 37 degrees C under standard lymphocyte culture conditions. Freezing the cells for 10 days in a cryoprotective medium preserved the ability of the cells to prolong graft survival. In fact, freeze-thawed cells were more effective than fresh cells. Extension of the ALS-bone marrow treatment protocol to human transplantation is expected to be facilitated by frozen and short-term refrigerated storage of the donor bone marrow.

Mouse skin graft prolongation with donor strain bone marrow and anti-lymphocyte serum: surface markers of the active bone marrow cells.

The survival of C3H/HeJ skin grafts on B6AF1 mice treated with anti-lymphocyte serum (ALS) can be significantly prolonged by the injection of the host with C3H/HeJ bone marrow. Although the prolongation is apparently due at least in part to the ultimate presence in the host of specific suppressor cells of donor origin, little is known about the nature of the cells in the marrow inoculum that are responsible for this effect. The present investigation was undertaken to characterize surface markers of the active bone marrow cells. Marker-positive populations were either depleted and enriched by panning techniques or depleted by killing with specific antibody and complement, and then were assayed for their ability to prolong graft survival. Cells that were adherent to anti-Ia-coated plates extended graft survival only slightly better than did treatment with ALS alone, whereas nonadherent (Ia-depleted) cells, as well as cells treated with anti Ia and complement, retained good prolonging activity. Similarly, panning on anti-immunoglobulin (Ig)-coated plates produced an active, Ig+-depleted population and an inactive adherent population, and killing of Thy-1+ cells with antibody and complement did not compromise the ability of the bone marrow inoculum to prolong graft survival. Complement receptor-positive (EAC+) and Fc gamma receptor-positive cells (EA+) were separated by panning on plates coated with sheep erythrocytes/antibody/complement and erythrocytes/7S antibody respectively. Adherent, EAC+-enriched cells were only slightly active, whereas the nonadherent, EAC-depleted population was fully active in graft prolongation. However, both Fc gamma R+ (EA+)-enriched and depleted populations were active, with the enriched fraction producing significantly better prolongation than the depleted population. Thus, the bone marrow cells that can prolong skin graft survival in ALS-treated mice appear to be Ia-, Thy-1+, largely complement receptor negative, and Ig-, but are largely positive for Fc gamma receptors.

Responses to preoptic temperature manipulation in the awake and hibernating marmot.

Responses of normothermic and hibernating marmots to manipulations of the preoptic-hypothalamic temperature (TPO) were studied. Independent variables included alteration of TPO and, during normothermia, room temperature. Hibernation occurred at an ambient of 6 degrees C. Dependent variables include brain, subdermal, and surface temperatures, heart rate, and behavioral, electromyographic cortical, and hippocampal responses. Although normothermic autumn marmots displayed most of the usual mammalian responses to alterations of the TPO, evidence of effective dermal vasomotion was not obtained. Single episodes of water drinking accompanied prolonged raising of the TPO; sleep was not elicited. During hibernation, effective central thermoregulation was not apparent until 3 or 4 days had elapsed. After this, thermoregulation was readily demonstrable in response to both raising and lowering the TPO. The apparent open-loop gains (OLG) for rise in body temperature after lowering of the TPO showed an exponential increase in value at lower prestimulus body temperatures. It was postulated that this could be explained on the basis of the recruitment of cold-sensitive neurones, which in turn would provide an explanation for the hypothesized "alarm temperature."