Canine bone marrow-derived mesenchymal stromal cells suppress alloreactive lymphocyte proliferation in vitro but fail to enhance engraftment in canine bone marrow transplantation.

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4 dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

Differential endocrine responses to infant odors in common marmoset (Callithrix jacchus) fathers.

Olfactory cues can exert priming effects on many mammalian species. Paternally experienced marmosets, Callithrix jacchus, exposed to direct isolated olfactory contact with their own infant's scent show rapid decreases in testosterone levels within 20 min, whereas paternally inexperienced males do not. The following study tests whether there is a differential steroid response to exposure of infant scent from dependent infants (own and novel) and independent infants (own and novel). We examined the serum levels of estradiol, estrone, testosterone, dihydrotestosterone (DHT), and combined estrogens and androgens in eight male marmosets 20 min after exposure to isolated infant scent. Testosterone and androgen levels combined were significantly lower with exposure to own infant scent than a novel infant scent when the infants were at a dependent age but not at an independent age. Estrogen levels elevated significantly in response to own infant scent when the infants were at a dependent age but not at an independent age. These results suggest that marmoset fathers are more responsive to priming cues from related infants and hormonal responses from fathers are greatest when the infant is at a dependent age.

Iterative development and pilot testing of an intervention fidelity monitoring plan for the enhanced, electronic health record-facilitated pragmatic clinical trial: Implications for training and protocol integrity.

Fidelity monitoring is the degree to which a clinical trial intervention is implemented as intended by a research protocol. Consistent implementation of research protocols supported with extant fidelity monitoring plans contribute rigor and validity of study results. Fidelity monitoring plans should be comprehensive yet practical to accommodate the realities of conducting research, particularly a pragmatic clinical trial, in dynamic settings with heterogeneous patient populations. The purposes of this paper are to describe the (1) iterative development and implementation of protocols for intervention fidelity monitoring, (2) pilot testing of the fidelity monitoring plan, (3) the identification of interventionist training deficiencies, and (4) opportunities to enhance protocol rigor for a cancer symptom management intervention delivered through the electronic health record patient portal and telephone as part of a complex, multi-component pragmatic clinical trial to uncover training deficits and bolster protocol integrity. The intervention focuses on prominent symptoms reported among medical oncology patients including sleep disturbance, pain, anxiety, depression, low energy (fatigue) and physical function. In this pragmatic trial, the role of interventionist is a registered nurse symptom care manager (RN SCM). A three-part fidelity monitoring plan with checklists audit: Part-1 RN SCM role training activities in research components, clinical training components, and protocol simulation training; Part-2 RN SCM adherence to the intervention core components delivered over the telephone; and Part-3 maintenance of adherence to core intervention components. The goal is ≥ 80% adherence to components of each of the three checklists. An initial pilot test of the fidelity monitoring plan was conducted to evaluate the checklists and the RN SCM adherence to core protocol components. RN SCM skills and training deficits were identified during the pilot phase, as were opportunities to improve protocol integrity. Overall, approximately 50% of the audited RN SCM telephone calls had ≥80% fidelity to the core components. There remains on-going need for RN SCM training and skill building in action planning. The content presented in this paper is intended to begin to fill the gap of fidelity monitoring plans for complex interventions tested in pragmatic clinical trials and delivered remotely in an effort to strengthen protocol integrity.

Delaying DLA-haploidentical hematopoietic cell transplantation after total body irradiation.

Exposure to accidental or deliberate radiation poses a threat to public health, proving lethal at higher doses in large part because of deleterious effects on marrow. In those cases, allogeneic hematopoietic cell transplantation (HCT) might be required to restore marrow function. Most radiation accident victims will have HLA-haploidentical relatives who could serve as HCT donors. Here, we assessed in a canine HCT model the total body irradiation (TBI) doses after which transplants might be required and successful engraftment would be possible. In an attempt at mimicking the logistical problems likely to exist after radiation accidents, 4-, 8- or 10-day intervals were placed between TBI and HCT. To keep the experimental readout simple, no graft-versus-host disease (GVHD) prevention was administered. All dogs transplanted after a 4-day delay following 700 or 920 cGy TBI successfully engrafted, whereas virtually all those given 450 or 600 cGy rejected their grafts. Transplant delays of 8 and 10 days following 920 cGy TBI also resulted in successful engraftment in most dogs, whereas a delay of 8 days after 700 cGy resulted in virtually uniform graft failure. The time courses of acute GVHD (aGVHD) and rates of granulocyte recovery in engrafting dogs were comparable among dogs regardless of the lengths of delay. In other studies, we showed that most dogs not given HCT survived 700 cGy TBI with intensive supportive care, whereas those given 800 cGy TBI and higher died with marrow aplasia. Thus, DLA-haploidentical HCT was successful even when carried out 4, 8, or 10 days after TBI at or above radiation exposures where dogs survived with intensive care alone.

Determinants of physical activity among women treated for breast cancer in a 5-year longitudinal follow-up investigation.

OBJECTIVE: To estimate the 5-year trajectory of physical activity among women with breast cancer, and to evaluate biopsychosocial variables (health status, physical symptoms, health-related quality of life (HRQL), depressive symptoms, and social support), measured soon after breast cancer diagnosis, as predictors of the 5-year trajectory. METHODS: Women diagnosed with Stage II or III regional breast cancer (n=227), surgically treated and awaiting the start of adjuvant therapy completed baseline assessments of medical, psychological, and behavioral functioning. Follow-up evaluations were conducted every 4 months during the first year and every 6 months during the subsequent 4 years (12 assessments total during the 5-year study). Mixed-effects modeling was utilized to estimate the baseline level of physical activity as well as rate of change over time. Measures of physical health status, HRQL, depressive symptoms, and social support were included as predictors of the physical activity trajectory. RESULTS: A curvilinear pattern of change in physical activity was evident over the 5-year follow-up (p=0.002). Physical activity increased gradually during the first 18 months, then declined steadily over the subsequent 42 months. Poor physical health, depressive symptoms, and lower emotional HRQL were associated with less physical activity. Higher family support was associated with a slower decline in physical activity in the latter 42 months of the study. CONCLUSIONS: Emotional HRQL following diagnosis with breast cancer appears to be important for sustaining physical activity in the first 1-2 years following diagnosis. Physical activity interventions among breast cancer survivors should address depressive symptoms early in the course of treatment.

In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs.

We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K. All three dogs had stable gene marking and donor chimerism before receiving a course of O(6) -benzylguanine (O(6) BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) between days 210 and 589 after transplantation. One to four doses led to a marked increase in gene marking in all dogs. Furthermore, the transduced cells conferred chemoprotection and prevented severe neutropenia. Our results suggest that drug resistance gene therapy is feasible and safe in the nonmyeloablative transplantation setting.

Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, or foamy virus.

Recent advances have allowed for improved retrovirus-mediated gene transfer, and therapeutic benefits have been described in patients. These successes have shown the potential of hematopoietic stem cell (HSC) gene therapy, but treatment-related leukemia and benign expansion of gene-modified clones have shifted the attention toward safety. The delayed onset of adverse events in gene therapy clinical trials emphasizes the importance of long-term integration site studies in large animal models. We have addressed safety by characterizing the genomic location of 555 integration sites of the three most commonly used integrating retroviral vectors, that is, gammaretrovirus, lentivirus, and foamy virus, in long-term repopulating cells from dogs. Gammaretroviral integrants showed the most significant frequency of occurrence very close (<2.5 kb) to transcription start sites, but a substantial portion of all three retroviral integrants were within 50 kb. Importantly, gammaretroviral integrants were found more frequently in and near proto-oncogenes, suggesting this retroviral system may be the most prone to adverse gene activation. These data suggest that gammaretroviral vectors may have the highest intrinsic risk, but also emphasize that no vector system can be defined as "safe" based solely on integration profile.

Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model.

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

Using snacks high in fat and protein to improve glucoregulatory function in adolescent male marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus), a laboratory nonhuman primate, is a well-known model of several human diseases and conditions, but the nutritional needs of these animals are not fully understood. Here we describe a 4-mo controlled study in which we increased the dietary fat and protein of subadult male common marmosets by using healthy snacks. Six male marmosets received their normal diet (control), and an additional 6 were given their normal diet supplemented daily with a 14-kcal snack. Cashews and waxworms were used as the snack, given their high-fat content. Although body weight did not differ between the 2 groups, only control male marmosets showed increased chest circumferences over the course of the study. Glucoregulatory function remained consistent in the snack-fed marmosets, whereas control animals had progressed toward higher insulin. Other indices of glucoregulation indicated significant differences in adiponectin and the cortisol:cortisone ratio between the 2 groups, but no differences in lipid concentration were detected. Therefore, the most notable difference attributable to the snack feeding was improved glucoregulation. Because the snacks we used had a high proportion of unsaturated compared with saturated fat, we suggest that these healthy high-fat-high-protein snacks provide an important contribution to the nutrition of this laboratory species. This study also demonstrates the utility of marmosets as a model for understanding the implications of dietary fats in humans.

Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation.

BACKGROUND: It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function. METHODS: The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb. RESULTS: One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells. CONCLUSION: These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

Improved short-term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells.

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols. Here, we compared the short-term engraftment of lentivirally and gammaretrovirally transduced canine allogeneic DLA-matched littermate cells. A reduced conditioning regimen of 400 cGy total body irradiation was used in preparation for clinical studies. Two dogs received a graft of gammaretrovirally transduced CD34-selected cells. CD34(+) cells were prestimulated for 30 h and then exposed twice to concentrated RD114 pseudotype vector. Three dogs received lentivirally transduced CD34-selected cells. Cells were transduced overnight with concentrated VSV-G pseudotype lentiviral vector. The animals in the lentiviral group showed a significantly faster granulocyte recovery. VNTR analysis 40-50 days after transplantation revealed higher donor chimerism for the lentiviral group compared to the retroviral group. These data suggest that short lentiviral transduction protocols may be superior to extended gammaretroviral transduction protocols with respect to engraftment potential of transduced CD34(+) hematopoietic repopulating cells.

Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy.

Incorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)-transduced autologous CD34(+)-selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O(6)BG and temozolomide resulted in a multilineage increase in gene-modified repopulating cells with marking levels of greater than 98% in granulocytes. MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in conjunction with O(6)BG and temozolomide in clinical trials.

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.

The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

Pharmacologically regulated in vivo selection in a large animal.

The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.