Pharmacologic, toxicologic, and marrow transplantation studies in dogs given succinyl acetone.

A novel immunosuppressant, succinyl acetone (4,6-dioxoheptanoic acid), was studied in dogs. Results with bolus intravenous injections at doses ranging from 50 to 1600 mg/kg showed dose-dependent alpha and beta half-lives, ranging from 30 to 80 min and 7 to 27 hr, respectively. Results suggested that continuous i.v. infusion was necessary to maintain constant plasma levels. Four dogs were given 9.2 Gy total-body irradiation and autologous marrow transplants along with continuous i.v. infusion of succinyl acetone at 50, 100, 200, or 400 mg/kg/day for 21 days, and all four had rapid, sustained hematopoietic engraftment. However, two of the four dogs receiving 200 and 400 mg succinyl acetone/kg/day, respectively, developed bilateral hind-limb ataxia, with histologically confirmed cerebellar lesions in the dog given the higher dose, thus establishing a potential dose-limiting neurotoxicity. Prevention of graft-versus-host disease was studied in recipients of allogeneic marrow. Dogs were given 9.2 Gy TBI, followed by hematopoietic grafts from unrelated DLA-nonidentical or DLA-haploidentical littermate dogs. Succinyl acetone was given as continuous infusion for 21 days after transplant at doses of 100-300 mg/kg/day. Starting succinyl acetone on the day of marrow infusion in four dogs failed to prevent rapid onset of acute GVHD, and dogs survived no longer than controls. Starting succinyl acetone 3 days before transplant delayed the onset of acute GVHD and prolonged survival significantly compared with that of dogs not given postgrafting immunosuppression (P = 0.008); survival was comparable to that in previously reported dogs given either methotrexate or cyclosporine as postgrafting immunosuppression (P = 0.88 and 0.99, respectively). Seven of the sixteen allogeneic recipients developed evidence of neurotoxicity during succinyl-acetone infusion. Neurological dysfunctions were manifested by hind-limb ataxia and posterior paresis. In conclusion, succinyl acetone significantly delayed the onset of GVHD and prolonged survival of DLA-nonidentical marrow graft recipients but did not induce graft-host tolerance and was associated with dose-limiting neurotoxicity.     

What role for FTY720, a novel immunosuppressive agent, in canine nonmyeloablative hematopoietic stem cell transplantation?

BACKGROUND: Stable mixed donor/host hematopoietic chimerism was almost uniformly achieved in dogs given 200 cGy total body irradiation (TBI) before, and a short course of immunosuppression after, transplantation of marrow from dog leukocyte antigen-identical littermates, but was transient when the TBI dose was decreased to 100 cGy. Here, we examined whether stable engraftment could be achieved in five dogs given FTY720 (days -5 and -4), followed by 100 cGy TBI, dog leukocyte antigen-identical marrow grafts, and mycophenolate mofetil/cyclosporine. RESULTS AND CONCLUSIONS: Although all five dogs showed initial engraftment, four dogs rejected their grafts within 11 weeks, whereas one dog was euthanized on day 17 due to enteritis. This was not different from the control dogs not given FTY720 (P=0.32). Thus, FTY720 failed to enhance allogeneic engraftment in this model, perhaps due to in vivo T-cell depletion of the graft resulting from sequestration of donor lymphocytes in host central lymphoid tissues.     

Transplantation of DLA-compatible and incompatible fetal liver hematopoietic cells in dogs

Requirements for sustained engraftment of fetal liver hematopoietic stem cells were evaluated in 45 dogs. Pretransplant preparative treatment with total-body irradiation, 14.7 Gy, permitted engraftment of DLA-compatible fetal liver cells. Radiation alone was inadequate in DLA-haploidentical or DLA-mismatched transplants; none of 5 dogs had engraftment. Addition of cyclosporine facilitated engraftment. The combination of 14.7 or 16.1 Gy total-body irradiation (TBI) and cyclosporine allowed engraftment in 15 of 19 (78%) dogs receiving DLA-histoincompatible grafts and 11 Gy TBI plus cyclosporine allowed engraftment in 4 of 10 dogs. Restoration of granulopoiesis and thrombopoiesis was rapid; recovery of lymphocytes was relatively delayed, especially in recipients of incompatible fetal liver cells. Cumulative one-year survival was decreased in recipients of incompatible grafts due to early posttransplant infections. These data suggest that fetal liver transplantation is a potential approach in patients who lack an HLA-identical donor for bone marrow transplantation.     

Hematopoietic stem cells from the marrow of mice treated with Flt3 ligand are significantly expanded but exhibit reduced engraftment potential

BACKGROUND: Hematopoietic stem cells (HSC) can be significantly expanded by hematopoietic growth factors. Flt3 ligand (FL) is a hematopoietic growth factor that induces proliferation and mobilization of HSC into the peripheral blood. We previously reported that FL-mobilized HSC exhibit superior engraftment potential. The engraftment potential of FL-expanded HSC in the bone marrow compartment has not been evaluated. In this study, we investigated the effect of in vivo administration of FL on the engraftment potential of HSC expanded in the marrow. METHODS: B10.BR (H-2k) donor mice were treated for 10 days with 10 microg of FL per day. Partially conditioned allogeneic B10 (H-2b) recipients received whole bone marrow. Purified HSC (c-Kit+/Sca1+/lin-) from the marrow were also transplanted in ablated syngeneic B10.BR recipients. RESULTS: FL treatment significantly expanded HSC in the marrow compartment. The absolute number of T cells and granulocytes were unchanged whereas dendritic cells, facilitating cells, and HSC were significantly increased in the bone marrow of donor mice treated with FL compared with untreated mice. Mice conditioned with 700 cGy and transplanted with FL-treated allogeneic bone marrow showed a significantly lower rate of engraftment (14%) compared with recipients of bone marrow from untreated mice (100%). Syngeneic recipients transplanted with 500, 1000, 2000, or 3000 purified HSC from FL-treated donors also showed reduced long-term survival compared with mice transplanted with HSC from untreated donors. Cell cycle analysis revealed that significantly more bone marrow HSC were in cycle after FL treatment compared with unmanipulated controls. CONCLUSION: These data show that FL treatment for 10 days induces proliferation of HSC but reduces the engraftment potential of HSC harvested from the marrow. The reduced syngeneic engraftment of HSC indicates that FL treatment induces intrinsic changes in HSC, resulting in failure of long-term engraftment or self-renewal despite no change in characteristic phenotype of HSC.     

The use of an anti-TCRalphabeta monoclonal antibody to control host-versus-graft reactions in canine marrow allograft recipients conditioned with low dose total body irradiation.

BACKGROUND: A limitation in the application of marrow transplantation has been complications related to the conditioning regimens that have been intensified to the point where organ toxicities have been common, resulting in morbidity and mortality. METHODS: A conditioning regimen consisting of low-dose total body irradiation (TBI*) was used to test whether postgrafting therapy with a monoclonal antibody (mAb) against the T cell receptor (TCR)alphabeta facilitated sustained engraftment of marrow from dog leukocyte antigen (DLA)-identical canine littermates. The anti-TCRalphabeta mAb 15.9D5 was selected for in vivo studies because it induced hyporesponsiveness to allogeneic stimulator cells in mixed leukocyte culture and was not mitogenic in vitro. RESULTS: When recipients of genotypically DLA-identical marrow were conditioned by the barely "lethal" dose of 450 cGy TBI alone, almost 60% of grafts failed (n=39). The remainder engrafted, either in the form of stable mixed donor/host or all donor hematopoietic chimerism. In contrast to results in controls, 5 of 6 dogs that were given, in addition, a loading dose of mAb 15.9D5 of 1 mg/kg on day -1, 450 cGy TBI on day 0, followed by mAb at 0.3 mg/kg/day until day +7, showed sustained engraftment (P=.058). To accomplish a comparable rate of engraftment in the absence of anti-TCRalphabeta antibody, 920 cGy TBI were needed for pretransplant conditioning. CONCLUSIONS: Results strongly suggested that in vivo administration of a mAb against TCRalphabeta prevented rejection of allogeneic marrow grafts in the setting of conditioning with a relatively nontoxic but otherwise suboptimal dose of 450 cGy TBI. In vivo administration of m Ab 15.9D5 was well tolerated without any noticeable side effects. The exact mechanism by which the mAb works in vivo is as yet poorly understood, but it does not involve CD3/TCR complex modulation or elimination of T cells from the circulation.     

Engraftment of DLA-nonidentical bone marrow facilitated by recipient treatment with anti-class II monoclonal antibody and methotrexate

Marrow transplants were carried out between unrelated DLA-nonidentical dogs. Recipients were prepared for transplantation by 9.2 Gy single-dose total-body irradiation (TBI) administered at 7 cGy/min. All recipient dogs received marrow, 4 X 10(8) cells/kg, and no buffy coat cells. In addition to TBI, 19 dogs were given anti-class II monoclonal antibody (mAb) 7.2, either alone or combined with a second anti-class II mAb, HB10a, at doses of 0.2 or 0.4 mg/kg/day on days -5 to 0 or -1 to +3. Seven had hemopoietic engraftment. Ten dogs were given, in addition to TBI, methotrexate (MTX), 0.4 mg/kg/day on days 1, 3, 6, and 11 after grafting, and six had engraftment. Ten dogs were given a combination of mAb 7.2 pregrafting and MTX after grafting, and 9 had evidence of engraftment. Among 7 dogs treated with the irrelevant mAb 31E6.4, only one had engraftment. Thus the administration of anti-class II mAb pregrafting and MTX postgrafting had a beneficial effect in overcoming resistance to histoincompatible marrow grafts. Possibly this effect is more pronounced when both approaches are combined.     

Combined immunosuppression with cyclosporine and methotrexate in dogs given bone marrow grafts from DLA-haploidentical littermates

We examined the effect of methotrexate (MTX) combined with cyclosporine (CSP) on the prevention of graft-versus-host disease (GVHD) in dogs. Ten recipients were prepared for grafting with 9 Gy of total-body irradiation and given marrow and buffy coat cells from littermate donors differing for one DLA haplotype (AA leads to AB or AB leads to AC). MTX (0.4 mg/kg) was given i.v. on days 1, 3, 6, and 11. CSP was given i.m. on days 0 to 7 and orally on days 8 to 100. The dose was 15 mg/kg/day on days 0 to 25, 10 mg/kg/day on days 26 to 50, 5 mg/kg/day on days 51 to 75, and 5 mg/kg every other day on days 75 to 100. All ten dogs had sustained engraftment. Three dogs died, one with pneumonia (day 57), one with pneumonia and GVHD (day 107), and one with convulsions (day 204). Seven dogs are surviving at 210 to 493 days, and all are complete chimeras; five are well and two have chronic GVHD that developed after immunosuppressive treatment had been stopped. These results with a combination of MTX and CSP as GVHD prophylaxis are superior to those obtained previously with MTX alone in dogs given marrow grafts from DLA-haploidentical littermates.     

Bone marrow transplantation in miniature swine. II. Effect of selective genetic differences on marrow engraftment and recipient survival

In order to study the effect of defined genetic differences on bone marrow transplantation in miniature swine, five different combinations of major histocompatibility complex (MHC)-matched and mismatched bone marrow transplants were performed. Eight of nine fully MHC-mismatched allogeneic bone marrow transplants failed to reconstitute, and one animal reconstituted briefly but then died quickly thereafter. Five of six class I-matched/class II-mismatched (g----c) bone marrow transplants engrafted, showed a skin rash typical of graft-versus-host (GVH) reaction, and died 3 weeks after the marrow transplantation. None of five class II-matched/class I-mismatched (g----d) transplants engrafted. Parental marrow transplants into F1 hosts engrafted and caused GVH skin rash, with survivals from 1 to 9 months (n = 5). Serologic typing of the F1 recipients of parental marrow showed only donor-type peripheral blood lymphocytes (PBL), suggesting complete marrow replacement. Conversely, F1 into parental marrow transplants showed no engraftment (n = 6). These results indicate that resistance to MHC-mismatched allogeneic bone marrow engraftment in swine represents a host response recognizing donor class I MHC differences. This response appears to interfere with engraftment of donor bone marrow cells despite host preparation with 900-1100 rads total-body irradiation. In the absence of donor MHC class I differences, engraftment was seen despite the existence of multiple non-MHC differences, and even in the presence of class II differences. Such engraftment also led to GVH, varying in intensity according to the strength of genetic disparity (i.e., worst in parent----F1 combination). These results suggest that miniature swine should provide an effective model for study of both GVH elimination (in the parent----F1 combination) and problems of engraftment (in the F1----parent combination), the two most important obstacles to clinical allogeneic transplantation.     

An anti-CD44 antibody does not enhance engraftment of DLA-identical marrow after low-dose total body irradiation

920 cGy total body irradiation (TBI) is adequate for consistently successful engraftment of marrow from dog leukocyte antigen (DLA)-identical littermates; however, the dose is inadequate to ensure a marrow graft from DLA-nonidentical unrelated donors. Such mismatched grafts are successful only after 1800 cGy, given in three fractions. While anti-T-cell reagents enhance engraftment of DLA-identical littermate marrow after 920 cGy, they fail to be effective in the DLA-nonidentical setting. However, a monoclonal antibody (mAb) to CD44, S5, was found to be very effective in enhancing engraftment of DLA-nonidentical marrow. The current study asked whether mAb S5 was also effective in the setting of DLA-identical littermate transplants. To this purpose, the TBI dose was lowered to 450 cGy, a dose after which 70% of such grafts failed. Four dogs were treated with antibody S5, 0.2 mg/kg on days −7 though −2 (per previously published protocol), given 450 cGy TBI followed by marrow grafts from their DLA-identical littermates. All four dogs rejected their grafts; two of these died from marrow aplasia, and two survived with endogenous marrow recovery. This result was not statistically significantly different from that in 17, historical (n = 5) and concurrent (n = 12), control dogs where 11 of 17 animals rejected. Even if ten experimental animals were transplanted and all six remaining dogs engrafted, the results still would not have been significantly different from control. This result is in contrast to the successful engraftment promoted by pretreatment with antibody S5 of DLA-nonidentical unrelated dogs, consistent with the notion that different host cells are involved in graft rejection in the two disparate histocompatibility settings.     

Engraftment following T cell-depleted bone marrow transplantation. III. Differential effects of increased total-body irradiation on semiallogeneic and allogeneic recipients

Three different doses of total-body irradiation (TBI) (1100, 1300, 1500 cGy) have been analyzed as conditioning regimens for semiallogeneic B6AF1 (H-2b X H-2a) and allogeneic A/J (H-2a) recipients of T cell-depleted C57BL6 (H-2b) bone marrow transplants. Recipient survival and engraftment of both donor erythrocytes and lymphocytes were examined in each group. The large majority of allogeneic mice prepared with 1100 cGy rejected their grafts, which resulted in poor survival (less than 30%); improved survival (up to 80%) and complete donor engraftment were noted as the TBI dose was increased. By contrast, survival in semiallogeneic B6AF1 recipients was independent of TBI dose and was greater than 80% in all groups. Outright failure of marrow grafts (less than 10% donor hematopoiesis) did not occur in these recipients, but mixed chimerism (simultaneous occurrence of both donor and host cells) was frequently observed at lower TBI doses. Complete (greater than 90%) donor engraftment was noted for erythrocytes but not for lymphocytes. Possible mechanisms accounting for these differences between semiallogeneic and allogeneic recipients of marrow transplants are discussed.     

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.     

Facilitation of engraftment of DLA-nonidentical marrow by treatment of recipients with monoclonal antibody directed against marrow cells surviving radiation

Past studies in dogs have suggested that marrow graft rejection was mediated by major histocompatibility complex (MHC) class II antigen-positive non-T cells that survived standard doses of total-body irradiation (TBI). We have now raised 4 monoclonal antibodies (mAbs) against marrow cells harvested 6 days after TBI. The mAbs are highly reactive (greater than 70%) with marrow cells surviving radiation and also bind strongly (greater than 50%) to normal marrow cells, lymphocytes, monocytes, and granulocytes. One of the mAbs (34-S3) reacted strongly with NK-like cells. In vitro treatment of marrow with mAb and rabbit complement (C') did not affect erythroid colony-forming unit (CFU-E) growth, whereas 2 of the 4 mAbs inhibited granulocyte-macrophage colony-forming unit (CFU-GM) growth, and all 3 mAbs tested suppressed autologous marrow engraftment. One of the mAbs, 69-S5 (IgG1), bound to a 95,000 dalton antigen. It crossreacted with human cells, but not with cells from Rhesus monkeys, baboons, and cats. We administered this mAb intravenously at 0.2 mg/kg/day on days -5 to 0 to dogs given 9.2 Gy TBI on day 0 followed by marrow grafts (less than or equal to 4 x 10(8) cells/kg) from DLA-nonidentical unrelated donors. Three of five dogs had sustained grafts. Increasing the dose of mAb ten-fold (2 mg/kg/day) resulted in graft failure (2 of 2 dogs). Treatment with a dose of 0.2 mg/kg/day from day -7 to -2 showed sustained engraftment in 7 of 10 dogs. This result is in contrast to sustained grafts in 3 of 36 dogs not given mAb, and in 1 of 7 dogs treated with an irrelevant mAb (P = 0.0002 and 0.04, respectively). We conclude that treatment of recipients with a mAb raised against marrow cells surviving radiation and not directed at major histocompatibility complex (MHC) class II antigens and NK-like cells can also facilitate engraftment of DLA-nonidentical canine marrow. These results may be relevant for the transplantation of HLA-incompatible marrow in man, particularly after in vivo T cell depletion, where graft failure is frequent.     

A partial conditioning approach to achieve mixed chimerism in the rat: depletion of host natural killer cells significantly reduces the amount of total body irradiation required for engraftment.

BACKGROUND: Mixed allogeneic bone marrow chimerism induces tolerance to solid organ grafts. Although we previously reported that partially ablative conditioning with 700 cGy of total body irradiation (TBI) is sufficient to allow for bone marrow engraftment in mice, we determined that a minimum of 1000 cGy was required in the rat. Because T cells and NK cells are critical in bone marrow graft rejection, our purpose was to examine whether targeting of radioresistant NK cells and/or T cells in the recipient hematopoietic microenvironment would reduce the TBI dose required for engraftment of allogeneic rat bone marrow. METHODS: Wistar Furth rats received either anti-NK3.2.3 monoclonal antibodies on days -3 and -2, anti-lymphocyte serum on day -5, a combination of both or no pretreatment. TBI was performed on day 0 and rats were reconstituted with 100x10(6) T cell-depleted bone marrow cells from ACI donors. RESULTS: Engraftment of T cell-depleted rat bone marrow was readily achieved in animals conditioned with 1000 cGy TBI alone (12/12) and the level of donor chimerism averaged 89%. At 900 cGy TBI alone only one of eight recipients engrafted. In striking contrast, 11 of 12 animals pretreated with anti-NK monoclonal antibodies and irradiated with 900 cGy showed donor chimerism at a mean level of 41%. No further enhancement of bone marrow engraftment could be achieved when recipients were pretreated with antilymphocyte serum alone or antilymphocyte serum plus anti-NK monoclonal antibodies. Mixed allogeneic chimeras exhibited stable multilineage chimerism and donor-specific tolerance to subsequent cardiac allografts. CONCLUSION: Specific targeting of radioresistant host NK cells allows for a significant reduction of the TBI dose required for allogeneic bone marrow engraftment.     

Marrow grafts between canine litter-mates homozygous or heterozygous for lymphocyte-defined histocompatibility antigens.

Hemopoietic grafts following 1,200 R of total body irradiation were carried out between canine littermates homozygous or heterozygous for lymphocyte defined (LD) antigens of the major histocompatibility complex (MHC). In all but five of the 25 pairs studied, LD homozygous dogs were also homozygous for serologically defined (SD) antigens of the MHC. Results of transplants were compared with previous results obtained in littermate pairs matched or mismatched for the MHC. Two groups of recipients were studied. Of 14 LD homozygous recipients in group 1 given grafts from LD heterozygous donors, 12 died between 8 and 193 days and two survived more than 238 and 627 days. The most frequent cause of death was graft-versus-host disease (GVHD). Survival was significantly shorter (P less than 0.005) than that of dogs given grafts from matched littermates and not different (P congruent to 0.5) from that of dogs given grafts from mismatched littermates. Survival of 11 LD heterozygous recipients in group 2 given grafts from LD homozygous donors was not different from that of dogs in group 1 (P congruent to 0.5). The results indicate that the LD loci detected by mixed leukocyte culture are not the principal determinants within the MHC that are responsible for GVHD.     

Immunoglobulin levels in dogs after total-body irradiation and bone marrow transplantation

The influence of total-body irradiation (TBI) and autologous or allogeneic bone marrow transplantation on serum immunoglobulin subclasses was determined in a dog model. Only IgG1 levels decreased after low-dose (+/- 4.5 Gy) TBI, but levels of all immunoglobulin classes fell after high-dose TBI (8.5 GyX1 or 2X6.0 Gy). After autologous bone marrow transplantation IgM levels were the first and IgE levels were the last to return to normal. After successful allogeneic bone marrow transplantation prolonged low IgM and IgE levels were found but IgA levels increased rapidly to over 150% of pretreatment values. A comparison of dogs with or without clinical signs or graft-versus-host disease (GVHD), revealed no differences in IgM levels. Dogs with GVHD had higher IgA but lower IgE levels. Dogs that rejected their allogeneic bone marrow cells showed significant early rises in IgE and IgA levels in comparison with dogs with GVHD. These results differ from the observations made on Ig levels in human bone marrow transplant patients. No significant differences in phytohemagglutinin stimulation tests were found between dogs with or without GVHD or dogs receiving an autologous transplant for the first four months after TBI and transplantation. An early primary or secondary involvement of humoral immunity in GVHD and graft rejection in dogs is postulated.     

ABO compatibility and acute graft-versus-host disease following allogeneic bone marrow transplantation

If ABO antigens/antibodies play any role in the pathogenesis of acute graft-versus-host disease (GVHD), one would expect the highest incidence of GVHD in recipients of minor ABO-mismatched grafts, followed by ABO-matched grafts, and the lowest incidence in major ABO-mismatched transplants. To test this hypothesis 174 patients receiving an HLA-identical allogeneic bone marrow transplant (BMT) for aplastic anemia (n = 32) or leukemia (n = 142) were analyzed for factors associated with acute GVHD. Variables analyzed included diagnosis, sex, age, blood group of donor and recipient, ABO compatibility, Rhesus compatibility, sex compatibility, number of bone marrow cells given at BMT, year of transplant, day of engraftment, and GVHD prophylaxis. We first carried out an exploratory contingency table analysis: minor ABO incompatibility was associated with a significantly higher risk of severe acute GVHD when compared with ABO-matched and major-ABO mismatched pairs (P = 0.003): 14/9, 57/67, and 5/22 patients developed, respectively, 0-I/II-IV acute GVHD in ABO major-mismatched, matched, and minor-mismatched pairs. Donors of group 0, (P = 0.06), older recipient's age (P = 0.08), fast engraftment (P = 0.03), and older donor's age (0.08) were also associated with a higher risk of GVHD. Recipient's ABO group, diagnosis, year of transplant, Rhesus group of donor or recipient, Rhesus compatibility, sex of donor or recipient, sex compatibility, and type of GVHD prophylaxis were not predictive of GVHD. A Cox multifactorial proportional hazards analysis confirmed that ABO matching was the single most significant factor associated with GVHD (P = 0.006). The cumulative incidence of GVHD grade II+ was 39%, 54%, and 82% for ABO major-mismatched, matched, and minor-mismatched pairs (P = 0.01). This study suggests that ABO antigens may play a role in the development of acute GVHD.     

同种异基因造血干细胞移植急性移植物抗宿主病小鼠模型的制作

目的 制作同种异基因造血干细胞移植急性移植物抗宿主病(GVHD)小鼠模型.方法 以C57BL/6( H-2b)小鼠为供者,Balb/c( H-2d)小鼠为受者,进行同种异基因骨髓移植.设立全身照射(TBI)对照组(4只)、GVHD组(10只)、单纯骨髓移植组(10只)及正常对照组(4只).TBI对照组仅进行致死性TBI,TBI后不进行骨髓移植;GVHD组于TBI前5d开始饮用含320 mg/L庆大霉素和250 mg/L红霉素的饮用水,移植当天以60Co γ射线行一次性TBI,总剂量8.0Gy,TBI后5h内每只小鼠经尾静脉输注C57BL/6小鼠骨髓细胞2×106个+脾细胞1×107个;单纯骨髓移植组预处理与GVHD组相同,每只小鼠经尾静脉输注C57BL/6小鼠骨髓细胞2×106个.移植后观察小鼠的精神状态、活动能力、体位改变、皮毛、体重和大便等,记录每只小鼠的存活时间,计算存活率,并绘制生存曲线.濒死小鼠的皮肤、肝脏、小肠和骨髓行病理检查.结果 TBI对照组小鼠的存活时间为(9.0±0.7)d,GVHD组为(32.0±3.2)d,单纯骨髓移植组为(17.5±1.6)d,3组间两两比较,存活时间的差异均有统计学意义(P＜0.01).TBI对照组病理检查显示造血功能衰竭.GVHD组于移植后第10～13天出现急性GVHD表现,其皮肤、肝脏和小肠组织的病理表现均符合Ⅰ～Ⅱ度急性GVHD改变,单纯骨髓移植组也于移植后第10～13天出现GVHD表现,但其GVHD表现和组织学改变明显轻于GVHD组,仅为0～Ⅰ度GVHD.结论 Balb/c小鼠经致死性TBI后移植同种异基因小鼠骨髓细胞+脾细胞可成功制作稳定的急性GVHD模型.     

Intensified postgrafting immunosuppression failed to assure long-term engraftment of dog leukocyte antigen-identical canine marrow grafts after 1 gray total body irradiation

BACKGROUND: Late graft rejection after conditioning with 1 Gy of total body irradiation (TBI) was consistently seen in historical dogs given two postgrafting immunosuppressive drugs. METHODS: Here, 16 dogs were given four different three-drug combinations of cyclosporine, mycophenolate mofetil, sirolimus, or methotrexate after 1 Gy TBI and dog leukocyte antigen-identical marrow grafts. In addition, we assessed the effects of TBI doses of 0.5, 1.0, 2.0, or 3.0 Gy, respectively, on immune functions in six dogs not given marrow grafts. RESULTS: All dogs showed initial engraftment, 13 rejected, and three had sustained grafts beyond 26 weeks. The dogs with durable grafts had received greater median numbers of nucleated marrow cells compared with the 13 dogs that rejected their grafts (6.14 vs. 3.6 x 10(8) per kg; P=0.03). In a Cox proportional hazard model, which included data from 16 historical dogs, each increase in transplanted marrow cell numbers by 1 x 10(8) per kg decreased the hazard ratio of rejection by 0.5. Decreasing percents of remaining CD3, CD4, and CD8 cells in peripheral blood and lymph nodes were observed with increasing TBI doses. Further, greater suppressions of B-cell- and T-cell-dependent production of IgM and IgG antibodies in response to sheep red blood cell injections were observed after 2 Gy compared with 1 Gy TBI. CONCLUSION: Overall, triple postgrafting immunosuppression after 1 Gy TBI was well tolerated but failed to prevent graft rejection in this model. In vivo radiation studies have shown higher numbers of remaining host lymphocytes and better T-cell-dependent antibody production after 1 Gy compared with 2 Gy TBI.     

Fast hematopoietic recovery after bone marrow engraftment needs physiological proximity of stromal and stem cells

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT1(1)) rats were either injected IV with syngeneic sex-mismatched 80 x 10(6) I-BM or transplanted with 80 x 10(6) V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.