Safety and efficacy of a nonmyeloablative pretransplant conditioning regimen using total lymphoid irradiation with volumetric modulated arc therapy in healthy dogs: A pilot study.

Allogeneic hematopoietic cell transplantation (HCT) has been an effective treatment for human patients with haematological malignancies (Baron & Storb, 2006; Bair et al., 2020; Copelan et al., 2019). However, the optimal pretransplant conditioning treatment is unclear in canine allogeneic HCT. This pilot study aimed to evaluate the safety and efficacy of total lymphoid irradiation (TLI) with volumetric modulated arc therapy (VMAT) for a nonmyeloablative HCT conditioning. Six healthy dogs were treated with 8 or 12 Gy TLI using VMAT. Haematological and physical changes were recorded over 8 weeks. To assess the effect of peripheral lymphocyte condition, lymphocyte subset and proliferative ability were examined. At the end of the experiment, necropsy was performed. All dogs showed mild-to-moderate neutropenia and thrombocytopenia, and these haematological changes resolved spontaneously. One dog treated with 8 Gy TLI developed transient cutaneous infection. No major complication was seen in the other seven dogs. Myelocytes and erythroblast cytopenia of bone marrow were detected in two dogs treated with 12 Gy TLI. This study is the first report of TLI using VMAT in dogs, and results suggest that this regimen is a feasible nonmyeloablative treatment.

Developments and translational relevance for the canine haematopoietic cell transplantation preclinical model.

The development of safe and reliable haematopoietic cell transplantation (HCT) protocols to treat human patients with malignant and non-malignant blood disorders was highly influenced by preclinical studies obtained in random-bred canines. The surmounted barriers included recognizing the crucial importance of histocompatibility matching, establishing long-term donor haematopoietic cell engraftment, preventing graft-vs-host disease and advancing effective conditioning and post-grafting immunosuppression protocols, all of which were evaluated in canines. Recent studies have applied the tolerance inducing potential of HCT to solid organ and vascularized composite tissue transplantation. Several advances in HCT and tolerance induction that were first developed in the canine preclinical model and subsequently applied to human patients are now being recruited into veterinary practice for the treatment of malignant and non-malignant disorders in companion dogs. Here, we review recent HCT advancements attained in the canine model during the past 15 years.

JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Sensitivity of spermatogonia to irradiation varies with age in pre-pubertal ram lambs.

Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers to <30% of controls and fewer tubules contained differentiated germ cells. Lesser expression of spermatogonial genes, VASA and UCHL-1, was observed in the 15 Gy group. In Group 2, only 12 Gy treated tubules contained fewer spermatogonia. Knowledge of these subtle differences between age groups in the effect of irradiation doses on spermatogonia or differentiated germ cell numbers and the duration of recovery of spermatogonia numbers after irradiation will aid the timing of germ cell transplants into prepubertal recipient lambs.

Allogeneic ovarian transplantation using immunomodulator preimplantation factor (PIF) as monotherapy restored ovarian function in olive baboon.

PURPOSE: Allogeneic ovarian transplantation may be an alternative in the future to oocyte donation in women with premature ovarian failure. The objectives of this study were to (a) evaluate allotransplantation feasibility for restoration of ovarian function and (b) assess efficacy of synthetic preimplantation factor (PIF) monotherapy as sole immune-acceptance regimen. METHODS: This is an experimental animal study using non-human primates (Papio anubis). Allogeneic orthotopic ovarian tissue transplantation was performed in two female olive baboons. PIF was administered as a monotherapy to prevent immune rejection and achieve transplant maintenance and function. Subjects underwent bilateral oophorectomy followed by cross-transplantation of prepared ovarian cortex. Postoperatively, subjects were monitored for clinical and biochemical signs of graft rejection and return of function. Weekly blood samples were obtained to monitor graft acceptance and endocrine function restoration. RESULTS: Postoperatively, there were no clinical signs of rejection. Laboratory parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine) did not indicate organ rejection at any stage of the experiment. Initially, significant loss of follicles was noticed after grafting and serum follicle-stimulating hormone (FSH) and E2 levels were consistent with ovarian failure. Seven months after transplantation, one animal exhibited recurrence of ovarian endocrine function (perineal swelling, E2 rise, FSH decrease, and return of menstruation). CONCLUSIONS: Organ rejection after allogeneic ovarian transplantation was prevented using PIF as monotherapy for the first time and no side effects were recorded. The study suggests the clinical feasibility of ovarian allotransplantation to obtain ovarian function.

Intussusception in canine recipients of hematopoietic cell grafts and surgical correction.

Intussusception is a common complication after canine hematopoietic cell transplantation (HCT). The present study was undertaken to evaluate predisposing factors of intussusception and to test whether intussusception can be managed surgically during the period immediately after HCT. We determined the incidence of intussusception after HCT was performed in 325 canine recipients (autologous, n = 43; allogeneic, n = 282) during the interval from January 2002 to May 2005. Intussusception was diagnosed in 16 of 325 dogs (4.9%). Intussusception was not significantly associated with the dose of irradiation, source of hematopoietic graft, use of immunosuppressive agents, gender, or age at transplant. A group of 9 of the affected dogs underwent small-bowel resection after diagnosis, and 7 were managed without surgical intervention. Despite complicating factors such as gastrointestinal toxicity and low neutrophil and platelet counts induced by the marrow conditioning regimen and the use of immunosuppressive agents, successful surgical management of intussusception was achieved in 6 of 9 dogs, as compared with successful management of 0 of 7 without surgery. Intussusception did not recur after surgical intervention in any dog. Recent HCT and post-transplant immunosuppressive therapy are not absolute contraindications to surgical intervention for intussusception in canine recipients of HCT.

Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.

We have previously demonstrated, using chimeric resistant MRL/lpr mice, that a fractionated total body irradiation (FTBI) (5 Gy x 2 with a 4 h interval on the day before allogeneic bone marrow transplantation (BMT)) is the best conditioning regimen for the treatment of autoimmune diseases in radiosensitive MRL/lpr mice. In the present study, using various standard strains of mice (not radiosensitive mice), we explore the best protocol for irradiation (doses and intervals) as the conditioning regimen for allogeneic BMT. Recipient mice were exposed to various irradiation regimens: a single total body irradiation (TBI) of 9.5 or 12 Gy and FTBI of (5+5) Gy to (7+7) Gy with a 1 to 24 h interval. The method generally utilized for humans ((2+2) Gy with a 4 h interval for 3 days (total 12 Gy)) was also used. One day after the last irradiation, donor BMCs from BALB/c, C3H, or C57BL/6 (B6) mice were transplanted into C3H or B6 mice. The irradiation protocol of (2+2) Gy for 3 days was found to be insufficient to enable the complete removal of recipient immunocompetent cells, since donor-reactive T cells were observed in the recipient spleens and many recipient-type NK and CD4(+) cells were also detected in the recipient hematolymphoid tissues. In all the combinations, the highest survival rate was achieved in the recipients irradiated with (6+6) or (6.5+6.5) Gy with a 4 h interval. In the surviving mice, the hematolymphoid tissues had been fully reconstituted with donor cells.