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.

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.

First case of Mycobacterium haemophilum infection in an AIDS patient in Italy.

Mycobacterium haemophilum, a strongly acid- and alcohol-fast bacillus belonging to the group of non-tuberculous mycobacteria was first described in 1978 as the cause of cutaneous ulcerating lesions in a woman with Hodgkin's disease. Infection due to M. haemophilum is rare but increasing in prevalence in immnunosuppressed subjects, particularly in patients with acquired immunodeficiency syndrome (AIDS) patients. The skin is the most common site of infection with erythematous or violaceous papules and/or nodules that are usually painless at first, but some elements develop into abscesses or ulcers that can become very painful. The incidence of M. haemophilum is unknown, but cases of infection have been reported in Australia, Canada, the United States, France, Israel, the United Kingdom and Taiwan; to date no cases have been reported in Italy, thus the case reported here is apparently the first one observed in our country.

Evaluation of a rapid immunochromatographic test for the serologic diagnosis of tuberculosis in Italy.

OBJECTIVE: To determine specificity, sensitivity and predictive values of a rapid immunochromatographic assay (ICT tuberculosis) for the diagnosis of tuberculosis (TB) in an Italian clinical setting, and to identify tentative new guidance for the interpretation of test results. METHODS: The ICT tuberculosis test is an immunochromatographic test based on the detection of IgG antibodies directed against five highly purified antigens secreted by Mycobacterium tuberculosis during active growth. Sera from 60 patients with active pulmonary (48 sputum smear-positive and six sputum smear-negative cases) and extrapulmonary (six cases) TB were obtained. Personal, anamnestic and clinical data were investigated and recorded for each patient. The control groups comprised 156 subjects: 40 healthy individuals, half of them Mycobacterium bovis BCG-vaccinated, and 116 patients with mycobacterial diseases other than TB (five cases), with nonmycobacterial lung diseases (30 cases), with nonmycobacterial nonlung diseases (30 cases), with nonmycobacterial diseases and rheumatoid factors positivity (30 cases), and with asymptomatic HIV infection (21 cases). For 21 individuals the test was simultaneously performed with both serum and whole blood sample. Each positive result of the ICT test was reported with regard to the number (1-4), position (A, B, C, D) and color intensity (+ to ++++) of the evidenced lines in order to assess the quality of the antibody response. RESULTS: The overall sensitivity and specificity were 56.7% and 90.4%, respectively. The sensitivity for pulmonary TB patients was 61.1% (66.7% for smear-positive and 16.7% for smear-negative cases) and 16.7% for extrapulmonary TB patients. The difference between ICT results in pulmonary TB patients and control subjects was statistically significant (P < 0.0001). The analysis of the positive ICT tests revealed that samples with strong color intensity (>/=++) and specific antibodies bound to antigens immobilized on line D were significantly more frequent in TB patients than in controls (P = 0.001 and P= 0.027, respectively). ICT test results with the presence of at least three visible lines were more often observed in the TB patients than in controls, although not reaching statistical significance (P = 0.052). No difference was observed between the results of the ICT test performed both on serum and whole blood sample. CONCLUSIONS: The ICT tuberculosis test was confirmed to be rapid and easy to perform without requiring special equipment, both on serum and whole blood sample. Our data, in accordance with those obtained in a previous study conducted in extra-European countries, confirmed higher sensitivities for the smear-positive TB patients than for the smear-negative TB patients, and for pulmonary TB patients than for the extrapulmonary TB patients. Data obtained on the quality of antibody response in the ICT positive samples, might be used to improve the performance of the test.

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.

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.

Resolution of recurrent perianal condylomata acuminata by topical cidofovir in patients with HIV infection.

Anogenital condylomata acuminata are the most frequent clinical manifestation of genital human papillomavirus (HPV) infection. Association between human immunodeficiency virus (HIV) and HPV infections is frequent (range: 26-60% in males). Topical cidofovir (a nucleotide analogue antiviral drug active against a broad range of DNA viruses) is a potential treatment for anogenital warts in immunocompromised patients. We treated three HIV-infected patients with HPV perianal condylomas with topical 1% cidofovir in flexible collodion once a day for 2 weeks. The treatment resulted in complete clearance of the HPV lesions. The patients experienced mild transient erythema without any other side-effects. None of the patients relapsed during the 10-14-month follow-up period.

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.

Tropical pulmonary eosinophilia. Report of a case.

Tropical pulmonary eosinophilia (TPE) is an unusual manifestation of filarial infection, most commonly found in South-East Asia and caused by immunologic hyperresponsiveness to Wuchereria bancrofti and Brugia malayi. This report concerns a case of TPE in a 25-year-old Indian male who had been living in Italy for two years and was admitted to hospital with chest pain. Diethylcarbamazine therapy proved effective in rapidly eliminating symptoms and pulmonary abnormalities, as well as normalizing of laboratory findings.