Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model.

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15 mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n = 7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n = 8) or 60 minutes (IBM60; n = 7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.

Graft-versus-Host Disease in a Dog After Reduced-intensity Hematopoietic Stem Cell Transplantation from a DLA-identical Littermate.

BACKGROUND: Graft-versus-host disease (GvHD) is an adverse effect following hematopoietic stem cell transplantation (HSCT) in humans. Dogs represent a key model organism for the development of treatment protocols for HSCT. However, detailed descriptions of canine GvHD and its treatment are rare. Herein we describe the development of canine GvHD and therapeutic intervention. MATERIALS AND METHODS: A female Beagle received an allogeneic HSCT from a dog leukocyte antigen-identical littermate (conditioning with 4.5 Gy total body irradiation; immunosuppression with cyclosporine A). RESULTS: GvHD developed at day +52 and was treated with methylprednisolone, cyclosporine A, antibiotics, antiviral medication and analgesics. The dog initially responded to the treatment but GvHD relapsed twice. Within one week after discontinuation of glucocorticoid, GvHD recurred resulting in inevitable euthanasia of the animal. CONCLUSION: GvHD represents a life-threatening disease after HSCT in canines. Immediate therapeutic treatment is indicated and even a successful initial treatment response does not necessarily prevent GvHD recurrence.

Low Radiation Dose and Low Cell Dose Increase the Risk of Graft Rejection in a Canine Hematopoietic Stem Cell Transplantation Model.

The canine hematopoietic stem cell transplantation (HSCT) model has become accepted in recent decades as a good preclinical model for the development of new transplantation strategies. Information on factors associated with outcome after allogeneic HSCT are a prerequisite for designing new risk-adapted transplantation protocols. Here we report a retrospective analysis aimed at identifying risk factors for allograft rejection in the canine HSCT model. A total of 75 dog leukocyte antigen-identical sibling HSCTs were performed since 2003 on 10 different protocols. Conditioning consisted of total body irradiation at 1.0 Gy (n = 20), 2.0 Gy (n = 40), or 4.5 Gy (n = 15). Bone marrow was infused either intravenously (n = 54) or intraosseously (n = 21). Cyclosporin A alone or different combinations of cyclosporine A, mycophenolate mofetil, and everolimus were used for immunosuppression. A median cell dose of 3.5 (range, 1.0 to 11.8) total nucleated cells (TNCs)/kg was infused. Cox analyses were used to assess the influence of age, weight, radiation dose, donor/recipient sex, type of immunosuppression, and cell dose (TNCs, CD34(+) cells) on allograft rejection. Initial engraftment occurred in all dogs. Forty-two dogs (56%) experienced graft rejection at median of 11 weeks (range, 6 to 56 weeks) after HSCT. Univariate analyses revealed radiation dose, type of immunosuppression, TNC dose, recipient weight, and recipient age as factors influencing long-term engraftment. In multivariate analysis, low radiation dose (P < .001) and low TNC cell count (P = .044) were identified as significant independent risk factors for graft rejection. Peripheral blood mononuclear cell chimerism ≥30% (P = .008) and granulocyte chimerism ≥70% (P = .023) at 4 weeks after HSCT were independent predictors of stable engraftment. In summary, these data indicate that even in low-dose total body irradiation-based regimens, the irradiation dose is important for engraftment. The level of blood chimerism at 4 weeks post-HSCT was predictive of long-term engraftment in the canine HSCT model.

Everolimus in combination with mycophenolate mofetil as pre- and post-transplantation immunosuppression after nonmyeloablative hematopoietic stem cell transplantation in canine littermates.

The mammalian target of rapamycin inhibitor everolimus (RAD001) is a successfully used immunosuppressant in solid-organ transplantation. Several studies have already used RAD001 in combination with calcineurin inhibitors after hematopoietic stem cell transplantation (HSCT). We investigated calcineurin inhibitor-free pre- and post-transplantation immunosuppression of RAD001 combined with mycophenolate mofetil (MMF) in a nonmyeloablative HSCT setting. After nonmyeloablative conditioning with 2 Gy total body irradiation, 8 dogs received HSCT from dog leukocyte antigen-identical siblings. Immunosuppressives were given at doses of 1.5 mg RAD001 twice daily from day -1 to +49, then tapered until day +56, and 20 mg/kg MMF from day 0 to +28, then tapered until day +42. An historical cyclosporin A (CsA)/MMF regimen was used in the control group. All dogs engrafted. Median platelet nadir amounted in all dogs to 0 × 10(9)/L (median, day +10; duration <50 × 10(9)/L, 22 days) and median leukocyte nadir was 1.0 × 10(9)/L (range, .1 to 2.5 × 10(9)/L; median, day +13). Eventually, 5 of 8 (63%) animals rejected their grafts. Two dogs died of infections on day +19 and +25. Pharmacokinetics of RAD001 and MMF showed median trough levels of 19.1 (range, 10.5 to 43.2) µg/L and .3 (.1 to 1.3) mg/L, respectively. The median area under the curve was 325 (range, 178 to 593) µg/L × hour for RAD001 and 29.6 (range, 7.9 to 40.5) ng/L × hour for MMF. All dogs developed clinically mucosal viral infections during the clinical course. Compared with the control group, the level of toxicities for RAD001/MMF increased in all qualities. Combined immunosuppression of RAD001 and MMF after nonmyeloablative HSCT is associated with significant toxicities, including a prolonged platelet recovery time as well as increased infections compared to the CsA/MMF regimen.