Organ Doses Associated with Partial-Body Irradiation with 2.5% Bone Marrow Sparing of the Non-Human Primate: A Retrospective Study.

A partial-body irradiation model with approximately 2.5% bone marrow sparing (PBI/BM2.5) was established to determine the radiation dose-response relationships for the prolonged and delayed multi-organ effects of acute radiation exposure. Historically, doses reported to the entire body were assumed to be equal to the prescribed dose at some defined calculation point, and the dose-response relationship for multi-organ injury has been defined relative to the prescribed dose being delivered at this point, e.g., to a point at mid-depth at the level of the xiphoid of the non-human primate (NHP). In this retrospective-dose study, the true distribution of dose within the major organs of the NHP was evaluated, and these doses were related to that at the traditional dose-prescription point. Male rhesus macaques were exposed using the PBI/BM2.5 protocol to a prescribed dose of 10 Gy using 6-MV linear accelerator photons at a rate of 0.80 Gy/min. Point and organ doses were calculated for each NHP from computed tomography (CT) scans using heterogeneous density data. The prescribed dose of 10.0 Gy to a point at midline tissue assuming homogeneous media resulted in 10.28 Gy delivered to the prescription point when calculated using the heterogeneous CT volume of the NHP. Respective mean organ doses to the volumes of nine organs, including the heart, lung, bowel and kidney, were computed. With modern treatment planning systems, utilizing a three-dimensional reconstruction of the NHP's CT images to account for the variations in body shape and size, and using density corrections for each of the tissue types, bone, water, muscle and air, accurate determination of the differences in dose to the NHP can be achieved. Dose and volume statistics can be ascertained for any body structure or organ that has been defined using contouring tools in the planning system. Analysis of the dose delivered to critical organs relative to the total-body target dose will permit a more definitive analysis of organ-specific effects and their respective influence in multiple organ injury.

Filgrastim for the treatment of hematopoietic acute radiation syndrome.

The U.S. Food and Drug Administration (FDA) recently approved Neupogen(®) (filgrastim) for the treatment of patients with radiation-induced myelosuppression following a radiological/nuclear incident. It is the first medical countermeasure currently approved by the FDA for this indication under the criteria of the FDA "animal rule". This article summarizes the consequences of high-dose radiation exposure, a description of the hematopoietic acute radiation syndrome (H-ARS), the use of hematopoietic growth factors in radiation accident victims and current available treatments for H-ARS with an emphasis on the use of Neupogen in this scenario.

Pegfilgrastim administered in an abbreviated schedule, significantly improved neutrophil recovery after high-dose radiation-induced myelosuppression in rhesus macaques.

Conventional daily administration of filgrastim is effective in reducing the duration of severe neutropenia and enhancing survival following lethal radiation, myelosuppressive cytotoxic therapy or myeloablation and stem cell transplantation. A sustained-duration form of filgrastim, pegfilgrastim has significantly simplified scheduling protocols after chemotherapy-induced neutropenia to a single injection while maintaining the therapeutic effectiveness of daily administration of filgrastim. We examined the ability of a single or double (weekly) administration of pegfilgrastim to significantly improve neutrophil recovery in a rhesus macaque model of severe radiation-induced myelosuppression. Animals were exposed to potentially lethal 6 Gy total-body X radiation. After irradiation all animals received supportive care and were administered either pegfilgrastim at 300 µg/kg on day 1 or day 1 and day 7 post exposure, or filgrastim at 10 µg/kg/day initiated on day 1 post exposure and continued daily through neutrophil recovery. Pharmacokinetic parameters and neutrophil-related values for duration of neutropenia, neutrophil nadir, time to recovery to an absolute neutrophil count ≥500/µL or ≥2000/µL, and days of antibiotic support were determined. Effective plasma concentrations of pegfilgrastim were maintained in neutropenic animals until after the onset of hematopoietic recovery, which is consistent with neutrophil-dependent properties of elimination. Administration of pegfilgrastim at day 1 and day 7 was most effective at improving neutrophil recovery compared to daily administration of filgrastim or a single injection of pegfilgrastim on day 1, after severe, radiation-induced myelosuppression in rhesus macaques.

ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs--threshold doses for tissue reactions in a radiation protection context.

This report provides a review of early and late effects of radiation in normal tissues and organs with respect to radiation protection. It was instigated following a recommendation in Publication 103 (ICRP, 2007), and it provides updated estimates of 'practical' threshold doses for tissue injury defined at the level of 1% incidence. Estimates are given for morbidity and mortality endpoints in all organ systems following acute, fractionated, or chronic exposure. The organ systems comprise the haematopoietic, immune, reproductive, circulatory, respiratory, musculoskeletal, endocrine, and nervous systems; the digestive and urinary tracts; the skin; and the eye. Particular attention is paid to circulatory disease and cataracts because of recent evidence of higher incidences of injury than expected after lower doses; hence, threshold doses appear to be lower than previously considered. This is largely because of the increasing incidences with increasing times after exposure. In the context of protection, it is the threshold doses for very long follow-up times that are the most relevant for workers and the public; for example, the atomic bomb survivors with 40-50years of follow-up. Radiotherapy data generally apply for shorter follow-up times because of competing causes of death in cancer patients, and hence the risks of radiation-induced circulatory disease at those earlier times are lower. A variety of biological response modifiers have been used to help reduce late reactions in many tissues. These include antioxidants, radical scavengers, inhibitors of apoptosis, anti-inflammatory drugs, angiotensin-converting enzyme inhibitors, growth factors, and cytokines. In many cases, these give dose modification factors of 1.1-1.2, and in a few cases 1.5-2, indicating the potential for increasing threshold doses in known exposure cases. In contrast, there are agents that enhance radiation responses, notably other cytotoxic agents such as antimetabolites, alkylating agents, anti-angiogenic drugs, and antibiotics, as well as genetic and comorbidity factors. Most tissues show a sparing effect of dose fractionation, so that total doses for a given endpoint are higher if the dose is fractionated rather than when given as a single dose. However, for reactions manifesting very late after low total doses, particularly for cataracts and circulatory disease, it appears that the rate of dose delivery does not modify the low incidence. This implies that the injury in these cases and at these low dose levels is caused by single-hit irreparable-type events. For these two tissues, a threshold dose of 0.5Gy is proposed herein for practical purposes, irrespective of the rate of dose delivery, and future studies may elucidate this judgement further.

Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas.

UNLABELLED: Side population (SP) cells defined by their ability to efflux Hoechst dye 33342 (Hst), demonstrate functional stem cell capabilities in adult murine tissues and may represent organ-specific stem cells. We examined adult human (Hu) and rhesus macaque (Rh) pancreatic tissue for the presence of SP cells. METHODS: Hu cadaver (n = 4) and Rh donor (n = 5) pancreata were dispersed with collagenase and separated by density gradient centrifugation to relatively enrich fractions for islet, ductal, and acinar tissue in human and islet and nonislet tissue in Rh. Single cell suspensions were incubated with varying Hst concentrations to determine optimal conditions for SP cell analysis. Cellular heterogeneity was assessed using a panel of monoclonal antibodies positive for hematopoietic and/or endothelial cells. RESULTS: Hu SP cells comprised approximately 0.12%, 0.08%, and 0.45% of the gated populations for Hu islet, ductal, and acinar fractions respectively. In Rh, 5.5% and 3.7% of the islet and nonislet fractions were identified as SP cells. FACS analysis of Hu pancreas-derived SP cells indicated that greater than 95% were CD45(-), and only 6% were CD34(+)CD45(-). A similar phenotype was detected in Rh pancreas-derived SP cell populations: greater than 70% were CD45(-) and less than 2% were endothelial lineage positive. CONCLUSIONS: SP cells are found in both islet- and nonislet-enriched fractions of the adult Hu and Rh pancreas. The majority of pancreatic SPs are CD45(-) and CD34(-), suggesting nonhematopoietic lineage. Further preclinical study is needed to establish the phenotype and functional role of adult tissue-specific versus tissue-resident stem cells.

Pegfilgrastim, a sustained-duration form of filgrastim, significantly improves neutrophil recovery after autologous marrow transplantation in rhesus macaques.

Daily administration of filgrastim decreases the duration of severe neutropenia in the clinical setting. A sustained-duration form of filgrastim, pegfilgrastim, significantly reduces scheduling protocols to a single injection per chemotherapy cycle while maintaining therapeutic efficiency. We examined the ability of a single injection of pegfilgrastim to significantly improve neutrophil recovery following autologous bone marrow transplantation (AuBMT) in rhesus macaques. On day 1, postmyeloablation (920 cGy x-irradiation) and AuBMT, animals received either 0.1% autologous serum for 18 consecutive days (n=13), or single doses of pegfilgrastim via the subcutaneous (s.c.) or intravenous (i.v.) route (300 or 100 micro g/kg), or a single dose of filgrastim at 300 micro g/kg via the s.c. or i.v. route, or filgrastim at 10 micro g/kg via the s.c. route (n=4) on a daily basis (range=days 12-17). Pharmacokinetic parameters and neutrophil recovery were assessed. A single dose of pegfilgrastim via the i.v. or s.c. route was as effective as daily filgrastim administration, resulting in significant improvement of neutrophil recovery after myeloablation and ABuMT. Effective pegfilgrastim plasma concentrations were maintained in neutropenic animals until after the onset of hematopoietic recovery. Enhanced pharmacokinetics in AuBMT cohorts are consistent with self-regulating, neutrophil-mediated clearance.

The impact of recent advances in immunology and cancer therapy on nuclear medicine.

The explosive expansion of knowledge in immunology in recent decades has already affected the research and practice of nuclear medicine in several ways. New hematopoietic cells have been isolated and their functions discovered, including hematopoietic stem cells and dendritic cells (DCs). Many new humeral factors have been found that have potent effects on cells, including cytokines, growth factors, and specialized proteins. Radiolabeled compounds are needed to follow the pharmacodynamics of the humeral factors and to follow the migration of mobile cells in animals and humans. In this article, only DCs, cytokines, and growth factors used clinically are discussed. DCs are essential for defense against infectious diseases. Autologous DCs cultured for a week and pulsed with tumor antigens have already proved highly immunogenic compared with other methods for activating cytotoxic T cells, and preliminary studies suggest that DCs are more potent for tumor cell killing than monoclonal antibodies. DCs, unfortunately, also play an important role in causing certain human diseases. In allograft transplants, residual donor DCs are responsible for the cellular rejection; if they could be eliminated, rejection could be prevented. These cells are also detrimental in rheumatoid arthritis, other autoimmune diseases, asthma, and chronic obstructive pulmonary disease. Cytokines such as interleukin-2 and such growth factors as granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, administered to patients with malignancies to alleviate the leukopenia of chemotherapy agents, frequently alter the tissue distribution of radiopharmaceuticals; these alterations may be confused with disease.

A single dose of pegylated leridistim significantly improves neutrophil recovery in sublethally irradiated rhesus macaques.

Leridistim, a member of the myelopoietin family of dual receptor agonists that binds interleukin-3 and G-CSF receptors, has been shown to enhance hematopoietic activity in rhesus monkeys above that observed with either cytokine alone or in combination. This study demonstrated the ability of a pegylated form of leridistim (peg-leridistim), administered s.c., as a single- or two-dose regimen separated by 4 or 7 days, to significantly improve neutrophil recovery following radiation-induced myelosuppression. Rhesus macaques were total body x-irradiated (250 kVp, TBI) to 600 cGy. Following TBI, two groups received peg-leridistim (n = 5) or leridistim (n = 4) at a dose of 600 microg/kg on day 1, while two other groups (both n = 4) received peg-leridistim at 200 microg/kg on day 1 and day 4, or day 1 and day 7. The irradiation controls (n = 7) received 0.1% autologous serum for 18 days. All peg-leridistim treatment schedules significantly improved all neutrophil-related parameters following TBI as compared with nontreated controls and were equivalent in effect when compared among themselves. Administration of a single high dose or two separate lower doses of peg-leridistim significantly improved neutrophil regeneration, in a manner equal to that of conventional daily or abbreviated every-other-day administration of leridistim in this nonhuman primate model of severe myelosuppression.

Leridistim, a chimeric dual G-CSF and IL-3 receptor agonist, enhances multilineage hematopoietic recovery in a nonhuman primate model of radiation-induced myelosuppression: effect of schedule, dose, and route of administration.

Leridistim is from the myelopoietin family of proteins, which are dual receptor agonists of the human interleukin-3 and G-CSF receptor complexes. This study investigated the effect of dosage, administration route, and schedule of leridistim to stimulate multilineage hematopoietic recovery in total body irradiated rhesus monkeys. Animals were x-irradiated on day 0 (600 cGy, 250 kVp) and then received, on day 1, leridistim s.c. in an abbreviated, every-other-day schedule at 200 microg/kg, or daily at 50 microg/kg, or i.v. daily or every-other-day schedules at 200 microg/kg dose. Other cohorts received G-CSF (Neupogen((R)) [Filgrastim]) in an every-other-day schedule at 100 microg/kg/day, or autologous serum (0.1%) s.c. daily. Hematopoietic recovery was assessed by bone marrow clonogenic activity, peripheral blood cell nadirs, duration of cytopenias, time to recovery to cellular thresholds, and requirements for clinical support. Leridistim, administered s.c. every other day, or i.v. daily, significantly improved neutrophil, platelet, and lymphocyte nadirs, shortened the respective durations of cytopenia, hastened trilineage hematopoietic recovery, and reduced antibiotic and transfusion requirements. A lower dose of leridistim administered daily s.c. enhanced recovery of neutrophil and platelet parameters but did not affect lymphocyte recovery relative to controls. Leridistim, a novel engineered hematopoietic growth factor administered at the appropriate dose, route and schedule, stimulates multilineage hematopoietic reconstitution in radiation-myelosuppressed nonhuman primates.

Pharmacodynamic model of topotecan-induced time course of neutropenia.

Pharmacodynamic measures of neutropenia, such as absolute neutrophil count at nadir and neutrophil survival fraction, may not reflect the overall time course of neutropenia. We developed a pharmacokinetic-pharmacodynamic model to describe and quantify the time course of neutropenia after administration of topotecan to children and to compare this with nonhuman primates (NHPs) as a potential preclinical model of neutropenia. Topotecan was administered as a 30-min infusion daily for 5 days, repeated every 21 days. As part of a Phase I Pediatric Oncology Group study, topotecan was administered at 1.4 and 1.7 mg/m(2)/day without filgrastim (POG), and at 1.7, 2, and 2.4 mg/m(2)/day with filgrastim (POG+G). In NHPs, topotecan was administered at 5, 10, and 20 mg/m(2)/day without filgrastim. A pharmacokinetic-pharmacodynamic model was fit to profiles of topotecan lactone plasma concentrations and neutrophil survival fraction from cycle 1 and used to calculate topotecan lactone area under the plasma concentration-versus-time curve from 0 to 120 h (AUC(LAC)) and the area between the baseline and treatment-related neutrophil survival fraction (ABC) from 0 to 700 h. The mean +/- SD neutrophil survival fraction at nadir for the POG, POG+G, and NHP groups was 0.12 +/- 0.09, 0.11 +/- 0.17, and 0.09 +/- 0.08, respectively (P > 0.05). The mean +/- SD for the ratio of ABC to AUC(LAC) for the POG and NHP groups was 1.02 +/- 0.38 and 0.16 +/- 0.09, respectively (P < 0.05). The model estimate of ABC and the ratio of ABC to AUC(LAC) in children and NHPs may better reflect sensitivity to chemotherapy-induced neutropenia.

Promegapoietin-1a, an engineered chimeric IL-3 and Mpl-L receptor agonist, stimulates hematopoietic recovery in conventional and abbreviated schedules following radiation-induced myelosuppression in nonhuman primates.

Promegapoietin-1a (PMP-1a), a multifunctional agonist for the human interleukin 3 and Mpl receptors, was evaluated for its ability to stimulate hematopoietic reconstitution in nonhuman primates following severe radiation-induced myelosuppression. Animals were total body x-irradiated (250 kVp) to 600 cGy total midline tissue dose. PMP-1a was administered s.c. in several protocols: A) daily (50 microg/kg) for 18 days; B) nine doses (5 microg/kg) every other day for 3 weeks; C) a single high dose (100 microg/kg) at 20 hours, or D) a single high dose (100 microg/kg) at 1 hour following TBI. The irradiation controls received 0.1% autologous serum for 18 consecutive days. Hematopoietic recovery was assessed by bone marrow clonogenic activity, peripheral blood cell nadirs, duration of cytopenias, time to recovery to cellular thresholds, and requirements for clinical support. PMP-1a, irrespective of administration schedule, significantly improved all platelet-related parameters: thrombocytopenia was eliminated, the severity of platelet nadirs was significantly improved, and recovery of platelet counts to > or =20,000/miccrol was significantly reduced in all PMP-1a-treated cohorts. As a consequence, all PMP-1a-treated cohorts were transfusion-independent. Neutrophil regeneration was augmented in all treatment schedules. Additionally, all PMP-1a-treated cohorts showed an improvement in red blood cell nadir and recovery. PMP-1a in conventional or abbreviated schedules induced significant thrombopoietic regeneration relative to the control cohort, whereas significant improvement in neutrophil recovery was schedule-dependent in radiation-myelosuppressed nonhuman primates.

Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRObeta.

SB-251353 is an N-terminal truncated form of the human CXC chemokine GRObeta. Recombinant SB-251353 was profiled in murine and rhesus monkey peripheral blood stem cell mobilization and transplantation models. SB-251353 rapidly and transiently mobilized hematopoietic stem cells and neutrophils into the peripheral blood after a single subcutaneous injection. Transplantation of equivalent numbers of hematopoietic stem cells mobilized by SB-251353 into lethally irradiated mice resulted in faster neutrophil and platelet recovery than stem cells mobilized by granulocyte colony-stimulating factor (G-CSF). A single injection of SB-251353 in combination with 4 days of G-CSF administration resulted in augmented stem and progenitor cell mobilization 5-fold greater than G-CSF alone. Augmented stem cell mobilization could also be demonstrated in mice when a single injection of SB-251353 was administered with only one-day treatment with G-CSF. In addition, SB-251353, when used as a single agent or in combination with G-CSF, mobilized long-term repopulating stem cells capable of hematopoietic reconstitution of lethally irradiated mice. In rhesus monkeys, a single injection of SB-251353 induced rapid increases in peripheral blood hematopoietic progenitor cells at a 50-fold lower dose than in mice, which indicates a shift in potency. These studies provide evidence that the use of SB-251353 alone or in combination with G-CSF mobilizes hematopoietic stem cells with long-term repopulating ability. In addition, this treatment may (1) reduce the number of apheresis sessions and/or amount of G-CSF required to collect adequate numbers of hematopoietic stem cells for successful peripheral blood cell transplantation and (2) improve hematopoietic recovery after transplantation.

Myelopoietin, an engineered chimeric IL-3 and G-CSF receptor agonist, stimulates multilineage hematopoietic recovery in a nonhuman primate model of radiation-induced myelosuppression.

Myelopoietins (MPOs) constitute a family of engineered, chimeric molecules that bind and activate the IL-3 and G-CSF receptors on hematopoietic cells. This study investigated the in vivo hematopoietic response of rhesus monkeys administered MPO after radiation-induced myelosuppression. Animals were total body irradiated (TBI) in 2 series, with biologically equivalent doses consisting of either a 700 cGy dose of Cobalt-60 ((60)Co) gamma-radiation or 600 cGy, 250 kVp x-irradiation. First series: On day 1 after 700 cGy irradiation, cohorts of animals were subcutaneously (SC) administered MPO at 200 microg/kg/d (n = 4), or 50 microg/kg/d (n = 2), twice daily, or human serum albumin (HSA) (n = 10). Second series: The 600 cGy x-irradiated cohorts of animals were administered either MPO at 200 microg/kg/d, in a daily schedule (n = 4) or 0.1% autologous serum (AS), daily, SC (n = 11) for 23 days. MPO regardless of administration schedule (twice a day or every day) significantly reduced the mean durations of neutropenia (absolute neutrophil count [ANC] < 500/microL) and thrombocytopenia (platelet < 20,000/microL) versus respective control-treated cohorts. Mean neutrophil and platelet nadirs were significantly improved and time to recovery for neutrophils (ANC to < 500/microL) and platelets (PLT < 20,000/microL) were significantly enhanced in the MPO-treated cohorts versus controls. Red cell recovery was further improved relative to control-treated cohorts that received whole blood transfusions. Significant increases in bone marrow-derived clonogenic activity was observed by day 14 after TBI in MPO-treated cohorts versus respective time-matched controls. Thus, MPO, administered daily was as effective as a twice daily schedule for multilineage recovery in nonhuman primates after high-dose, radiation-induced myelosuppression.

Myelopoietin, a chimeric agonist of human interleukin 3 and granulocyte colony-stimulating factor receptors, mobilizes CD34+ cells that rapidly engraft lethally x-irradiated nonhuman primates.

Myelopoietin (MPO), a multifunctional agonist of interleukin 3 and granulocyte colony-stimulating factor (G-CSF) receptors, was evaluated for its ability to mobilize hematopoietic colony-forming cells (CFC) and CD34+ cells relative to control cytokines in normal nonhuman primates. Additionally, the engraftment potential of MPO-mobilized CD34+ cells was assessed in lethally irradiated rhesus monkeys. Normal rhesus monkeys were administered either MPO (200 microg/kg/day), daniplestim (a high-affinity interleukin 3 receptor agonist) (100 microg/kg/day), G-CSF (100 microg/kg/day), or daniplestim coadministered with G-CSF (100 microg/kg/day each), subcutaneously for 10 consecutive days. The mobilization kinetics were characterized by peripheral blood (PB) complete blood counts, hematopoietic CFC [granulocyte-macrophage CFC (GM-CFC), megakaryocyte CFC (MK-CFC)], and the immunophenotype (CD34+ cells) of PB nucleated cells prior to and on day 3 to days 7, 10, 12, and 14, and at intervals up to day 28 following initiation of cytokine administration. A single large-volume leukapheresis was conducted on day 5 in an additional cohort (n = 10) of MPO-mobilized animals. Eight of these animals were transplanted with two doses of CD34+ cells/kg. A maximum 10-fold increase in PB leukocytes (white blood cells) (from baseline 7.8-12.3 x 10(3)/microL to approximately 90 x 10(3)/microL) was observed over day 7 to day 10 in the MPO, G-CSF, or daniplestim+G-CSF cohorts, whereas daniplestim alone stimulated a less than onefold increase. A sustained, maximal rise in PB-derived GM-CFC/mL was observed over day 4 to day 10 for the MPO-treated cohort, whereas the daniplestim+G-CSF, G-CSF alone, and daniplestim alone treated cohorts were characterized by a mean peak value on days 7, 6, and 18, respectively. Mean peak values for PB-derived GM-CFC/mL were greater for MPO (5,427/mL) than for daniplestim+G-CSF (3,534/mL), G-CSF alone (3,437/mL), or daniplestim alone (155/mL) treated cohorts. Mean peak values for CD34+ cells/mL were noted within day 4 to day 5 of cytokine administration: MPO (255/microL, day 5), daniplestim+G-CSF (47/microL, day 5), G-CSF (182/microL, day 4), and daniplestim (96/microL, day 5). Analysis of the mobilization data as area under the curve indicated that for total CFCs, GM-CFC, MK-CFC, or CD34+ cells, the MPO-treated areas under the curve were greater than those for all other experimental cohorts. A single, large-volume (3.0 x blood volume) leukapheresis at day 5 of MPO administration (PB: CD34+ cell/microL = 438 +/- 140, CFC/mL = 5,170 +/- 140) resulted in collection of sufficient CD34+ cells (4.31 x 10(6)/kg +/- 1.08) and/or total CFCs (33.8 x 10(4)/kg +/- 8.34) for autologous transplantation of the lethally irradiated host. The immunoselected CD34+ cells were transfused into autologous recipients (n = 8) at cell doses of 2 x 10(6)/kg (n = 5), and 4 x 10(6)/kg (n = 3) on the day of apheresis. Successful engraftment occurred with each cell dose. The data demonstrated that MPO is an effective and efficient mobilizer of PB progenitor cells and CD34+ cells, such that a single leukapheresis procedure results in collection of sufficient stem cells for transplantation and long term engraftment of lethally irradiated hosts.

Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning.

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

The combined administration of daniplestim and Mpl ligand augments the hematopoietic reconstitution observed with single cytokine administration in a nonhuman primate model of myelosuppression.

This study evaluated the ability of daniplestim, a high affinity interleukin 3 receptor agonist, to enhance the hematopoietic response of Mpl ligand (Mpl-L) administration in nonhuman primates following severe, radiation-induced myelosuppression. Rhesus monkeys were total body x-irradiated (TBI) to 600 cGy, midline tissue dose. Beginning on day 1 post-TBI, animals were s.c. administered daniplestim (100 microg/kg bid; n = 4), Mpl-L (10 microg/kg qd; n = 3), daniplestim (100 microg/kg bid) plus Mpl-L (10 microg/kg qd) (n = 4) or 0.1% autologous serum (AS) (n = 11) for 18 days. CBCs were monitored for 60 d after TBI. The duration of thrombocytopenia (platelet count; PLT <20,000/microl) was significantly decreased by the administration of daniplestim (6.5 d, p = .01), Mpl-L (3.0 d, p = .003) and the coadministered daniplestim/Mpl-L (1.3 d, p = .001) compared to controls (10.4 d). As monotherapy Mpl-L but not daniplestim significantly improved the PLT nadir (21,000/microl, p = .023 and 5,000/microl, p = .266, respectively) compared to the control (3,000/microl). The combined administration of daniplestim and Mpl-L significantly improved the PLT nadir (28,000/microl, p = .007) compared to both the control cohort (3,000/microl) and the daniplestim only cohort (5,000/microl, p = .043). Recovery of PLT to preirradiation values occurred earlier in the daniplestim only (d 21) or the daniplestim/Mpl-L cohorts (d 18) than in the Mpl-L only or control cohorts (d 28, d 29, respectively). The administration of daniplestim or Mpl-L alone neither shortened the duration of neutropenia (ANC<500/microl) compared to the controls (15.8 d, 16.0 d versus 16.2 d, respectively), nor improved the recovery time of neutrophils to baseline values (d 22, d 25, and d 23, respectively). The ANC nadir was significantly improved by daniplestim alone but not Mpl-L administration (76/microl, p = .001 and 50/microl, p = .093, respectively) compared to the controls (8/microl). Coadministration of daniplestim and Mpl-L significantly improved the ANC nadir (196/microl, p = .001) compared to either the AS- or the monotherapy-treated cohorts. Also the duration of neutropenia observed in the AS-controls (16.2 d) was significantly reduced in the daniplestim/Mpl-L cohort (10.8 d, p = .002). The combined administration of daniplestim and Mpl-L significantly improved hematopoietic recovery and further enhanced the stimulatory effect of cytokine monotherapy, as well as reducing clinical support requirements after radiation-induced bone marrow myelosuppression.

Therapy of radiation injury.

It is apparent from preclinical and clinical research to date that continued evaluation of new and alternative treatment strategies is required to eliminate the obligate periods of neutropenia and thrombocytopenia after acute high-dose irradiation. Future treatment strategies may involve new combinations of cytokines to affect hematopoietic stem cell proliferation and "engineered" cellular grafts to provide short-term in vivo expansion of neutrophils and platelets in an effort to bridge the cytopenic gap until endogenous or transplanted stem cells regenerate the hematopoietic and immune systems. Cytokine-mobilized peripheral blood and cord blood will provide alternative sources of allogeneic stem and progenitor cells in support of primary engraftment, delayed engraftment or secondary failure of the initial graft, as well as starting populations for various ex vivo expansion protocols. Further insights into the relative quality of stem cell populations and the factors that regulate their survival and self renewal, and the identification and roles of adhesion molecules in stem cell mobilization, engraftment, and interaction with the adult marrow microenvironment will provide the basis for future treatment strategies for the radiation-induced hematopoietic syndrome. As our ability to treat the hematopoietic syndrome improves, damage to other organ systems such as the skin, lung, and/or gastrointestinal tissue will emerge as dose-limiting. At the same time, the characterization of receptors for inflammatory cytokines, cytokine receptor antagonists, and anti-endotoxin antibodies has allowed significant insights into the mechanisms and pathogenesis of sepsis. However, translation of this knowledge into a treatment modality for septic patients is precluded by the lack of any clear-cut beneficial effect from the many clinical trials. The research and clinical results presented in this volume and recent conferences reflect the body of knowledge that will lead to further developments in assessment, prophylaxis, and treatment of radiation injuries in the areas of infectious disease and the hematopoietic, gastrointestinal, and cutaneous syndromes.

Combined administration of recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor enhances multilineage hematopoietic reconstitution in nonhuman primates after radiation-induced marrow aplasia.

This study compared the therapeutic potential of recombinant, native versus pegylated megakaryocyte growth and development factor (rMGDF and PEG-rMGDF, respectively), as well as that of the combined administration of PEG-rMGDF and r-methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF) on hematopoietic reconstitution after 700 cGy, 60Co gamma, total body irradiation in nonhuman primates. After total body irradiation, animals received either rMGDF, PEG-rMGDF, r-metHuG-CSF, PEG-rMGDF and r-metHuG-CSF or HSA. Cytokines in all MGDF protocols were administered for 21-23 d. Either rMGDF, PEG-rMGDF, or PEG-rMGDF and r-metHuG-CSF administration significantly diminished the thrombocytopenic duration (platelet count (PLT) < 20,000 per microliter)to o.25, 0, 0.5 d, respectively, and the severity of the PLT nadir (28,000, 43,000, and 30,000 per microliter, respectively) as compared with the controls (12.2 d duration, nadir 4,000 per microliter), and elicited an earlier PLT recovery. Neutrophil regeneration was augmented in all cytokine protocols and combined PEG-rMGDF and r-metHuG-CSF further decreased the duration of neutropenia compared with r-metHuG-CSF alone. These data demonstrated that the administration of PEG-rMGDF significantly induced bone marrow regeneration versus rMGDF, and when combined with r-metHuG-CSF significantly enhanced multilineage hematopoietic recovery with no evidence of lineage competition.

Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor.

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine-SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.