Erythrocytic Colonies Research Articles

Chimeric Antigen Receptor (CAR) T Cell Therapy for CD7-Positive Acute Myeloid Leukemia

Expression of CD7 is an adverse prognostic factor in both AML and myelodysplastic syndromes. Because CD7 is not constitutively expressed by mature myeloid cells or early myeloid progenitor cells, we explored the feasibility of targeting these malignancies using T cells expressing a CD7-directed chimeric antigen receptor (CAR7). Due to high expression of CD7 on normal T cells, expression of CAR7 induced substantial fratricide of T cells, preventing their expansion and long term survival. We therefore disrupted the CD7 gene in T cells using CRISPR/Cas9 prior to transduction with the CAR. Electroporation of the Cas9 protein with CD7-specific gRNA produced little toxicity to T cells, and the resulting CD7KOCAR7 T cells (henceforth CAR7 T cells) had no evident fratricide, expanding >300-fold within two weeks.Upon coculture with primary AML blasts, CAR7 T cells eliminated a mean 85% of malignant cells within 48h (Fig. A) and expanded 5-fold. Furthermore, CAR7 T cells effectively targeted primitive AML progenitor cells resulting in a mean >25-fold decrease in the number of leukemic colonies after a 5h coculture (Fig. B). CAR7 T cells demonstrated robust cytotoxicity against CD7+ AML cell lines eliminating >95% of malignant cells after 72h of coculture at a 1:40 effector-to-target ratio but had no activity against CD7-negative tumor cells. To test the protective effect in vivo, we established a mouse xenograft model of disseminated AML by intravenous injection of the CD7+ AML cell line KG-1a. A single injection of 2x10^6 CAR7 T cells produced a >2-log reduction in tumor burden and significantly extended median survival (65 days in control group; median survival not reached at 88 days for the CAR7 T treated animals, P=0.0025). In a second, more aggressive AML model using Kasumi-3 cells, we observed a 2-log reduction in tumor volume following administration of CAR7 T cells.CD7 may also be transiently expressed in normal early myeloid progenitors. To assess the off-tumor hematotoxicity of CAR7 T cells, we cocultured them with cord blood-derived hematopoietic progenitor cells and measured colony formation on a methylcellulose medium. We observed no differences in the numbers of granulocytic, monocytic and erythrocytic colonies after exposure to CAR7 T or control non-transduced T cells indicating a lack of toxicity to normal myeloid and erythroid progenitor cells (Fig. C). Similarly, we observed no reactivity of CAR7 T cells against peripheral monocytes. However, CAR7 T cells were cytotoxic to normal peripheral T- and NK-cells suggesting potential aplasia of these lymphocyte subsets subsequent to in vivo administration of the CAR7 T cells. We may be able to circumvent this toxicity, by infusing pathogen-directed CD7KO T cells from the same donor to repopulate the host, since CD7KO cells appear to retain TCR-mediated activity against a range of pathogens. Hence, CD7KO T cells expressing a CD7-directed CAR can expand without fratricide and have high activity against AML while sparing normal myeloid cells. These data support the feasibility of this approach for the targeted therapy of AML. [Display omitted] DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees.

Novel mutations in the EPO-R, VHL and EPAS1 genes in the Congenital Erythrocytosis patients

Congenital erythrocytosis (CE) can be classified as primary and secondary and 82 consecutive patients of erythrocytosis who were JAK-2 mutation negative, were further investigated. The genomic DNA was extracted from all the patients and the EPO-R, VHL, EGLN1 and EPAS1 genes were PCR amplified and sequenced. The sequence analysis showed (28/82) 34.14% patients had mutations. Among them, (19/28) 67.86% patients had mutations in exon 8 of EPO-R gene, of which six were novel missense mutations, p.(Gly418Ala), p.(Gly390Ala), p.(Ala411Thr), p.(Gly475Val), p.(Glu490Asp), p.(Glu362Gln) and three were novel frameshift mutations, p.(Glu336*), p.(Pro327Hisfs*68), p.(Gly479Alafs*37). All these EPO-R patients were heterozygotes and were forming endogenous erythrocyte colonies (EEC). Some patients (8/28) 28.57% had mutations in VHL gene, out of which 3 novel homozygous missense mutations in exon 1 of VHL gene, p.Gly80Asp, p.Gln107Glu and p.Gln113Glu, were identified. In addition, (1/28) 3.5% patients had one reported heterozygous missense mutation in exon 12 of EPAS1 gene p.Gly537Arg and one novel frameshift mutation p.(Ala553Glyfs*58). Further, in silico analysis indicated most of the mutations, probably, were damaging the protein structures, causing the CE in these patients. In this study the mutations in EPO-R and EPAS1 genes were identified for the first time in India.

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent.

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.

Novel Mutations in the <i>EPO-R, VHL</i> Genes and a Reported Mutation in <i>EPAS1</i> Gene in the Congenital Erythrocytosis Patients in a Southern State of India

Background: Congenital erythrocytosis (CE) can be classified as primary and secondary and 82 consecutive patients of erythrocytosis who were JAK-2 mutation negative, were further investigated. Methods: The genomic DNA was extracted from all the patients and the EPO-R, VHL, EGLN1 and EPAS1 genes were PCR amplified and sequenced. Findings: The sequence analysis showed 34.14% patients had mutations. Among them, 67.86% patients had mutations in exon 8 of EPO-R gene, out of which 7 patients showed novel missense mutations, p.(Gly418Ala), p.(Gly390Ala), p.(Ala411Thr), p.(Gly475Val), p.(Glu490Asp), p.(Glu362Gln) and three (3 out of 19) patients showed novel frameshift mutations, p.(Glu336*), p.(Pro327Hisfs*68), p.(Gly479Alafs*37), the rest being the reported ones, resulting in truncated protein formation. All these EPO-R patients were heterozygotes and were forming Endogenous Erythrocyte Colonies (EEC). Some patients (28.57%) had mutations in VHL gene, out of which 3 novel homozygous missense mutations in exon 1 of VHL gene, p.Gly80Asp, p.Gln107Glu and p.Gln113Glu, were identified. In addition, 3.5% patients had reported heterozygous missense mutation in exon 12 of EPAS1 gene p.Gly537Arg. Further, in silico analysis indicated most of the mutations, probably, were damaging the protein structures, causing the CE in these patients. Interpretation: Of the JAK-2-negative erythrocytosis patients in the southern state of India, 34.14% were seen to have CE, EPO-R alleles (67.86%) being the most frequently seen, followed by, VHL (28.57%) and EPAS1 (HIF-2α) (3.5%). Certain novel and reported mutations were identified. Nearly, one third of all CE patients presented with vascular thrombosis and CE could be an important etiological factor. Funding Statement: None. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was cleared by the Institutional Ethics Committee (IEC.NO: 966). A written consent was obtained from all the individuals studied.

Calcium-Infiltrated Biphasic Hydroxyapatite Scaffolds for Human Hematopoietic Stem Cell Culture.

Long-term in vitro expansion of hematopoietic stem cells (HSCs), while maintaining their functionality and multilineage differentiation potential, is still challenging. In this study, three-dimensional (3D) high-porosity hydroxyapatite (HA) foams have been designed to closely mimic the chemistry and physical structure of cancellous bone. Furthermore, calcium oxide was distributed in the HA ceramics to provide surface calcium ion release, hypothesizing that a local surface calcium gradient supports HSC localization and maintenance. Primary human HSCs and osteoblasts were cocultured for 6 weeks. Controls were cultured in two-dimensional dishes, while scaffold cultures were performed with calcium nitrate-infiltrated HA scaffolds and untreated HA scaffolds. Cells were analyzed for surface markers by flow cytometry, metabolic activity, and hematopoietic multilineage differentiation potential. The release of calcium into culture medium was also determined. The implementation of HA scaffolds had a positive effect on erythrocyte colony formation capacity of HSCs, with an increased osteoblast fraction observed when compared to control cultures without scaffolds. The presentation of scaffolds did not affect metabolic turnover when compared to control cultures. In conclusion, 3D open-porous HA scaffolds provide a bone-like structure and enable the long-term maintenance of primary HSCs.

Radioprotective effects of (-)-epigallocatechin-3-gallate on human erythrocyte/granulocyte lineages

Epigallocatechin-3-gallate (EGCg) is widely recognised as a powerful antioxidant and free radical scavenger. This study examined the radioprotective effects of EGCg on human granulopoiesis and erythropoiesis. Highly purified human CD34(+) haematopoietic stem/progenitor cells were prepared from human placental/umbilical cord blood. The cells were exposed to X rays at a dose rate of ∼1 Gy min(-1) and then cultured in a medium supplemented with either granulocyte colony-stimulating factor (G-CSF) or erythropoietin (EPO). EGCg (100 nM) was added to the culture immediately before or after X-irradiation. The concentration of 100-nM EGCg was determined in the authors' previous study. The number of granulocyte and erythrocyte colonies generated by X-irradiated CD34(+) cells decreased in a dose-dependent manner. Although EGCg addition yielded an ∼2-fold increase in the proliferation of each haematopoietic progenitor, no significant protective effect was observed in the surviving fraction of granulocyte progenitors (G-CSF alone: D(0)=1.06 Gy, n=1.14). However, EGCg addition before or after irradiation conferred a significantly higher protective effect on erythrocyte colony formation compared with the control (EPO alone: D(0)=0.66 Gy, n=1.56; EGCg (before): D(0)=0.43 Gy, n=5.48). EGCg addition before irradiation significantly improved the survival of erythroid progenitors subjected to radiation of <1 Gy. These results suggest that EGCg is more protective of erythropoiesis than granulopoiesis from radiation damage.

Preferential Inhibition of An Activated Form of Janus Kinase 2 (JAK2) by a Novel JAK2 Inhibitor, NS-018

AbstractAbstract 4107A somatic point mutation of Janus Kinase 2 (JAK2) tyrosine kinase (JAK2 V617F) has been shown to occur at a high frequency in myeloproliferative neoplasm (MPN) patients. JAK2 V617F is a constitutively activated kinase that activates the JAK/STAT signaling pathway and dysregulates cell growth and function. These findings suggest that the inhibition of aberrant JAK2 activation has a therapeutic benefit.Our novel JAK2 inhibitor, NS-018, is highly active against JAK2 with an IC50 value of less than 1 nM, and it has 30–50-fold selectivities for JAK2 over other JAK-family kinases such as JAK1, JAK3 and Tyk2. We determined the X-ray structure of JAK2 in complex with NS-018. An Asp-Phe-Gly (DFG) motif is located at the N-terminus of the activation loop and regulates ATP binding. The resolved X-ray structure showed that NS-018 bound to JAK2 in the “DFG-in” active conformation. A molecular modeling study indicated that NS-018 would hardly bind to JAK2 in the “DFG-out” inactive conformation. In accordance with the structural analysis, NS-018 preferentially suppressed the growth of bone-marrow cells expressing activated JAK2. Thus, NS-018 reduced in a dose-dependent manner the number of erythroid colony-forming units (CFU-E) derived from bone-marrow cells taken from JAK2 V617F transgenic mice, but had only a limited effect on the number of colonies from wild-type mice (Figure A). NS-018 had no effect on the number of granulocyte-macrophage colony-forming units (CFU-GM) from either mouse strain. Furthermore, NS-018 showed potent antiproliferative activity against Ba/F3 cells expressing JAK2 V617F with an IC50 value of <100 nM but showed only minimal cytotoxicity against most other hematopoietic and non-hematopoietic cell lines (IC50 >3 μ M). In a mouse Ba/F3-JAK2 V617F leukemia model, NS-018 significantly prolonged survival during repeated oral administrations at 6.25 mg/kg bid and reduced splenomegaly at doses as low as 1.5 mg/kg bid. NS-018 was well tolerated at dosages of more than 100 mg/kg bid. [Display omitted] In conclusion, NS-018 is a potent JAK2 inhibitor which preferentially inhibits an activated form of JAK2 and has potent in vitro and in vivo efficiency in preclinical studies. NS-018 is expected to be suitable for the treatment of MPN caused by aberrant JAK2 activation and its effectiveness will be verified by early-phase clinical investigations in the near future.JAK2 V617F preferential inhibition of erythrocyte colony growthBone-marrow cells were collected from femurs of JAK2 V617F transgenic mice and same-strain BDF1 wild-type mice. (a) To detect CFU-E colonies, cells were treated with NS-018 in semisolid methylcellulose containing erythropoietin (EPO) and cell clusters were counted after incubation for two days. (b) To detect CFU-GM colonies, cells were treated with NS-018 in semisolid methylcellulose containing EPO, interleukin-3 (IL-3), IL-6 and stem cell factor and colonies were counted on day 7. Disclosures:Nakaya:Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Sugahara:Nippon Shinyaku Co., Ltd: Employment. Horio:Nippon Shinyaku Co., Ltd: Employment. Niwa:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation

AbstractPrevious observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34+ progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters.

Budd-Chiari Syndrome and Portal Vein Thrombosis Associated with Myeloproliferative Disorders: Diagnosis and Management

The improvement observed in the prognosis of Budd-Chiari syndrome (BCS) since 1985 may be related to a better identification of underlying prothrombotic states. In a recent investigation of the currently identified prothrombotic disorders, one or several prothrombotic disorders were evidenced in 72% of patients with portal vein thrombosis (PVT) and 87% of patients with hepatic vein thrombosis. According to the different criteria used for their diagnosis, myeloproliferative disorders (MPDs) were reported in 25 to 65% of patients with splanchnic vein thrombosis (STV). In a single-center retrospective study of 128 patients with STV, clusters of abnormal megakaryocytes in bone marrow biopsy combined with endogenous erythrocyte colony formation were used as reference standards for a diagnosis of MPD. These two criteria allowed the diagnosis of MPDs at risk of aggravation in 25% (26 of 103) of patients with STV (41% in BCS; 23% in PVT). After a mean follow-up of 6.09 +/- 6.6 years, hematologic progression was confirmed in 23% of patients with the two criteria both positive, whereas neither progression nor features suggestive of MPD were observed in patients with the same concordant negative criteria. The combination of marked splenomegaly and platelet count > 200 x 10 (9)/L that was never observed in patients without MPD, constitutes a simple index for the suspicion of an underlying MPD, but the absence of an obvious splenic enlargement and normal or decreased platelet numbers may even be observed in patients with MPD-associated STV. In the absence of reliable indicators of the exact expansion of the myeloproliferative clone, the therapeutic management of STV-associated MPD is difficult to standardize. Owing to the suspected incidence on the prognosis of BCS of an associated MPD, the present watchful-waiting attitude proposed in MPD without obvious hematologic expression may be challenged.

Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)

AbstractThe erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the “nonstem,” “nonlymphoid” compartment (lineage marker [lin]–c-Kit+Sca-1–IL-7Rα–) into interleukin 3 receptor α negative (IL-3Rα–) and IL-3Rα+ subsets. Within IL-3Rα– but not IL-3Rα+ cells we have identified TER119–CD41–CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)–deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin–c-Kit+Sca-1–IL-7Rα–IL-3Rα–CD41–CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.

Ex Vivo Expansion of Hematopoietic Progenitors from CD34+-Selected Cells on Mouse Embryonic Fibroblast Feeders in a Non-Contact Co-Culture Using Matrigel or Fibronectin-Coated Inserts.

Adult pluripotent stem cells can be characterized as cells with both the capacity to self-renew and the ability to differentiate into distinct hematopoietic cell lineages and also into other tissue types. Because of the important course to stem cell research and clinical application the development of ex vivo culture systems is crucial for an effective expansion of these hematopoetic progenitor cells. After positive selection, CD34+-cells from peripheral G-CSF stimulated blood of healthy donors were cultured for 2 weeks in a serum-free medium with 2% of cord blood serum and the addition of early-acting cytokines (SCF, IL-3, Flt3-ligand, LIF, and TPO). The cells were grown in 6-well inserts on a feeder layer of mouse embryonic fibroblastic cells (MEF). Both cell types were separated from each other by a microporous membrane allowing only diffusion of soluble factors like cytokines but not cell migration. Additionally, the membrane was coated with Matrigel, a basement membrane matrix equivalent (MG), or with fibronectin (FN). Cultures were sampled at days 4, 7, 10 and 14 for cell count, colony-forming units-assay and immunophenotyping by flow cytometry. After two weeks of culture, the highest proliferation of stem cells was found in the control (medium without MEF, Matrigel and FN; 364-fold) followed by MEF- and FN-cultures (341- and 306-fold, respectively). The lowest clonal production was estimated in the cultures with MG (MG alone: 124,4-fold and MEF+MG: 48,2-fold, respectively). Examining the cells for colony-forming units the highest number of colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-GEM), burst-forming unit-erythrocyte (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit-erythrocyte (CFU-E) were found in cultures with MEF and MEF+FN. We found multipotential colonies (CFU-GEM and BFU-E) at the highest expansion rate with FN (13,3-fold) and MEF (8,2-fold) at the fourth day of culture and with both additives in a MEF+FN-culture at the seventh day (17,2-fold). In addition to these results, there were sporadic undifferentiated progenitor cell colonies in MEF+MG-culture up to the day 14, despite an initially lower growth rate than that on MEF alone. The percentages of CD34+-cells estimated by flow cytometry were downregulated continiously during the culture period. At day 7, the highest number of CD34+-cells was found in the cultures with MG (46,0%) and MEF+MG (42,0%). The maximum number of CD34+-cells expressing no CD38 but CD61 was found at day 4 in FN culture (20,4%) and MG culture (15,9 %), respectively. In summary, we recognized in cultures with MG as a factor similar to the natural microenvironment a correlation between the diminished proliferation rate of stem cells in favor of an increased generation of multipotential colonies combined with their delayed differentiation into granulocyte-macrophage and erythrocyte colonies. Appropiate feeder cells as well as matrix proteins like Matrigel or fibronectin may helpful in improving the ex vivo expansion of hematopoietic progenitor cells.

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

Small volume albumin administration protects against hemorrhagic shock-induced bone marrow dysfunction.

Unexpected immunomodulatory effects of colloids and crystalloids prompted an investigation of albumin's ability to prevent bone marrow (BM) suppression following trauma/hemorrhagic shock (T/HS: laparotomy + MAP 30 for 90 mins). In vitro: Normal rat BM was plated for granulocyte-macrophage (CFU-GM) and erythrocyte colony forming units (BFU-E) with 2% v/v plasma from sham (T/SS) or T/HS rats and albumin (2-8 mg/mL). In vivo: Male rats (n = 4/group) were subjected to T/SS or T/HS and resuscitated with shed blood and twice the volume as Lactated Ringer's (LR) or blood and 1, 2, or 3 mL of albumin (50 mg/mL). Bone marrow harvested 3 hours post-resuscitation was plated for CFU-GM and BFU-E. In vitro: T/HS plasma decreased both CFU-GM and BFU-E growth as compared with T/SS, whereas increasing doses of albumin showed dose-dependent improvement in progenitor growth (p < 0.05). In vivo: The suppression of BM red and white cell progenitor growth seen in T/HS+LR rats as compared with T/SS was fully prevented by as little as 1 mL of albumin (p < 0.05). Small doses of albumin fully restore CFU-GM and BFU-E to sham values. We postulate that the binding of circulating toxic factors by albumin may play a role in this prevention of T/HS-induced BM suppression.

Acute Trypanosoma cruzi infection is associated with anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia: reversal by nifurtimox treatment

In the present work we show that acute infection of C3H mice with the CL strain of Trypanosoma cruzi is characterized by an exponential growth of parasites and high mortality accompanied by anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia. Administration of nifurtimox, a trypanocydal drug currently in clinical use at different days postinfection, modulates parasitemia and prevents mortality. More importantly, none of blood and bone marrow alterations were observed in nifurtimox-treated animals when treatment was initiated early in infection, one or seven days postinoculation. The bone marrow alterations were characterized by a decrease in the total number cells as well in the number of megakaryoblasts and erythroblasts. Transfer experiments of bone marrow cells from infected mice to noninfected lethally irradiated recipients revealed a poor marrow-repopulating activity. The colony forming units-spleen assay confirmed the depression of committed clonal progenitors cells and revealed a decreased number of granulocyte/macrophage, megacariocyte and erythrocyte colonies. In summary, this is the first report showing that acute T. cruzi infection results in profound alterations of the hematopoietic system and that these alterations can be prevented by nifurtimox treatment.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF)–dependent in vitro and in vivo proliferation and differentiation of all hematopoietic progenitor cells in hGM-CSF receptor transgenic mice

To examine the relation between receptor expression and differentiation of hematopoietic cells, we produced transgenic mice that constitutively expressed the human granulocyte-macrophage colony stimulating factor (hGM-CSF) receptor at almost all stages of hematopoietic cell development. The high-affinity GM-CSF receptor is species specific, allowing analysis of the specific effects of hGM-CSF in our mouse model. Proliferation and differentiation of hematopoietic progenitor cells from transgenic mice were analyzed by means of methylcellulose colony-forming assay and in vivo treatment with hGM-CSF, respectively. We found that hGM-CSF supported various types of colonies, including granulocyte-macrophage, mast cell, megakaryocyte, blast cell, and mixed hematopoietic colonies, whereas mouse GM-CSF supported only granulocyte-macrophage colonies. In addition, hGM-CSF generated erythrocyte colonies in the absence of erythropoietin. Furthermore, in vivo administration of hGM-CSF to transgenic mice resulted in a dose-dependent increase in reticulocytes and white blood cells in the peripheral blood. The spleens of the mice showed gross enlargement, mainly caused by an increase of erythroid cells and their progenitors. Taken together, these results indicate that hGM-CSF receptor–mediated signals can support the growth of cells of all hematopoietic cell lineages if this receptor is present on the cell surface. This implies that the differentiation of hematopoietic progenitor cells is not determined by exogenous cytokine stimulation (instruction model) but by an intrinsic cell program in which cytokines simply select cells that express the appropriate receptor (stochastic model). (J Allergy Clin Immunol 1997;100:S79-86.)

Infection and replication of human cytomegalovirus in bone marrow stromal cells: effects on the production of IL-6, MIP-1alpha, and TGF-beta1.

We have investigated the mechanisms by which hematopoiesis is suppressed in patients suffering from human cytomegalovirus (HCMV) infections. Mixed populations of human bone marrow stromal and hematopoietic progenitor cells were inoculated with the Towne strain of HCMV to determine whether these populations could be infected and support HCMV replication. We found that the Towne strain of HCMV was capable of infecting and replicating in a mixed population of bone marrow stromal cells. We observed no significant alterations in bone marrow stromal cell proliferation or the production of IL-6, GM-CSF, soluble c-kit ligand and TNF-alpha following HCMV replication in either stimulated lipopolysaccharide (LPS) or unstimulated conditions. In samples of culture supernatants from LPS-stimulated HCMV-infected stromal cells, significant elevations in MIP-1alpha were observed. TGF-beta1 levels on the other hand exhibited two patterns following HCMV exposure; either TGF-beta1 levels decreased regardless of LPS stimulation or there was no effect. In addition, we observed that exposure to the Towne strain of HCMV resulted in significant inhibition of both granulocytic and erythrocytic colony formation in methylcellulose progenitor assays. Thus, both the direct effect of HCMV on hematopoietic progenitors as well as altered cytokine production by bone marrow stromal cells (including MIP-1alpha and TGF-beta1, but not IL-6) could contribute to hematopoietic failure during HCMV infection.

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted from delta TCS-1-positive T lymphocytes. At concentrations of 1.25-10 microM, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, of delta TCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.

Characterization of spleen colonies derived from mice with mutations at the w locus

Mice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU-S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony-derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI-1). Donor GPI-1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post-transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post-injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non-erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte-committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.

Immunization of simian immunodeficiency virus-infected rhesus monkeys with soluble human CD4 elicits an antiviral response.

Since the CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV), it has been suggested that a soluble truncated form of CD4 may compete with cell-surface CD4 for HIV binding and thus be of use in the therapy of AIDS. We have utilized the simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys to explore another possible therapeutic application of CD4 in AIDS--the use of recombinant soluble CD4 (rsCD4) as an immunogen. SIVmac-infected rhesus monkeys immunized with human rsCD4 developed not only an anti-human CD4 but also an anti-rhesus monkey CD4 antibody response. Coincident with the generation of this antibody response, SIVmac could not be isolated easily from peripheral blood lymphocytes and bone marrow macrophages of these animals. Furthermore, the decreased number of both granulocyte/macrophage and erythrocyte colonies grown from the bone marrow of these immunized monkeys rose to normal levels. These findings suggest that a modified human CD4 molecule serving as an immunogen might elicit an antibody response in man that could induce a beneficial therapeutic response in HIV-infected individuals.