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The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115+Gr-1neg monocyte precursor of resident tissue macrophages. CD115+Gr-1+ inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

11017Background: Tumor associated macrophages (TAM) are critical for immune evasion in many tumors. We hypothesized that combining G100, a TLR4 agonist that stimulates innate immunity and increases...

Purpose: Lysophosphatidic acid acyltransferase-β (LPAAT-β) is a transmembrane enzyme critical for the biosynthesis of phosphoglycerides whose product, phosphatidic acid, plays a key role in raf and AKT/mTor-mediated signal transduction. Experimental Design: LPAAT-β may be a novel target for anticancer therapy, and, thus, we examined the effects of a series of inhibitors of LPAAT-β on multiple human non–Hodgkin's lymphoma cell lines in vitro and in vivo. Results: We showed that five LPAAT-β inhibitors at doses of 500 nmol/L routinely inhibited growth in a panel of human lymphoma cell lines in vitro by >90%, as measured by [3H]thymidine incorporation. Apoptotic effects of the LPAAT-β inhibitors were evaluated either alone or in combination with the anti-CD20 antibody, Rituximab. The LPAAT-β inhibitors induced caspase-mediated apoptosis at 50 to 100 nmol/L in up to 90% of non–Hodgkin's lymphoma cells. The combination of Rituximab and an LPAAT-β inhibitor resulted in a 2-fold increase in apoptosis compared with either agent alone. To assess the combination of Rituximab and a LPAAT-β inhibitor in vivo, groups of athymic mice bearing s.c. human Ramos lymphoma xenografts were treated with the LPAAT-β inhibitor CT-32228 i.p. (75 mg/kg) daily for 5 d/wk × 4 weeks (total 20 doses), Rituximab i.p. (10 mg/kg) weekly × 4 weeks (4 doses total), or CT-32228 plus Rituximab combined. Treatment with either CT-32228 or Rituximab alone showed an approximate 50% xenograft growth delay; however, complete responses were only observed when the two agents were delivered together. Conclusions: These data suggest that Rituximab, combined with a LPAAT-β inhibitor, may provide enhanced therapeutic effects through apoptotic mechanisms.

PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study did not respond. Lysophosphatidic acid (LPA) is a phospholipid which mediates tumor cell migration and invasion. Recent studies have shown that inhibition of LPAAT-β inhibits both Ras/Raf/Erk and PI3K/Akt signaling cascades. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-β inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (ie, dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 lymphoma cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor (DFF) 45 is triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. Here we show that PS-341 does not induce phosphorylation of SEK-1, JNK, and c-Jun in DHL-4 cells, suggesting that it does not trigger a stress response. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggers swelling and cell membrane destruction in DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-β inhibitor CT-32615 triggers necrosis even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Background: Lysophosphatidic acid acyltransferase-β (LPAAT-β) catalyzes the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA), an essential component of the Ras pathway. In vascular smooth muscle cells, specific inhibitors of LPAAT-β interfere with the ras and PI3-kinase dependent signaling pathways thereby inducing growth arrest and apoptosis. We have previously shown that CT32228, a specific inhibitor of LPAAT-β, selectively inhibits the growth of CML progenitor cells in cultures without cytokines [ASH 2003, #2415]. Since Bcr-Abl is usually reactivated in cells resistant to the Abl kinase inhibitor imatinib, and Ras and PI3 kinase are downstream targets of Bcr-Abl, we hypothesized that a specific inhibitor of LPAAT-β may have activity against Bcr-Abl-positive CML with resistance to imatinib. Methods: Antiproliferative activity was assessed using the MTS-proliferation assay. Statistical analysis of combination studies (CT32228 + imatinib) was performed by applying the median effect method. Flow cytometry after staining with propidium iodide (PI) was performed to assess the cell cycle, co-staining with annexin allowed the detection of apoptotic cells. Signaling studies were performed by immunoblotting. Results: CT32228 inhibited the proliferation of Bcr-Abl expressing cell lines (AR230-s, K562, KCL22, Baf/BCR-ABL-s, 32DBCR-ABL, MO7p210) with IC50 concentrations between 23 and 105 nM. Similar IC50 values were obtained in lines expressing imatinib resistant BCR-ABL-mutants (Y253F/M351T/T315I/H396R) or amplified wild-type-BCR-ABL (Baf/BCR-ABL-r1, AR230-r), demonstrating the absence of significant cross-resistance. Combination studies with imatinib demonstrated additive to synergistic antiproliferative activity only in cell lines with residual sensitivity towards imatinib. FACS analysis of K562 cells treated with CT32228 showed accumulation of cells in G2/M as early as 6 h after start of treatment. After 24 h, 50% of cells were in G2/M and apoptosis was demonstrable with annexin/PI staining. Consistent with the G2/M arrest, multinucleated cells were evident microscopically at 24 h. Further analysis of apoptotic signalling pathways revealed activation of caspase-3 and PARP cleavage, while activation of caspase-8 and -9 was not demonstrated. In K562 cells treated with CT32228, Erk1/2 phosphorylation was abrogated, consistent with inhibition of mitogen activated kinase signalling as a result of decreased Ras signalling. In contrast phosphorylation of Akt was unaffected suggesting that inhibition of LPAAT-β does not interfere with the PI3K pathway in BCR-ABL-positive cells. Conclusion: CT32228 exerts antileukemic activity in imatinib-resistant cells without cross-resistance to imatinib and is synergistic in combination with imatinib. Cytotoxicity is mediated by cell cycle arrest in G2/M and induction of apoptosis. Interference with the Ras-pathway through MAPK-inhibition may play a key role in the antileukemic effect of CT32228. This data provides the framework for evaluation of CT32228 and related compounds to improve imatinib-based treatment of CML.

An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses. To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients. This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses. The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors. All of the helper viruses were in a single interference group. We have now shown that treatment of the M. dunni marker rescue assay cells with 5-iodo-2'-deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M. dunni endogenous virus (MDEV). Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium. MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect.

Mus dunni endogenous virus (MDEV) is transcriptionally inactive in cultured M. dunni cells but can be activated by treatment of the cells with either hydrocortisone or 5-iodo-2′-deoxyuridine (24). Activation can be easily measured because MDEV can package Moloney murine leukemia virus (Mo-MLV)-based retrovirus vectors. Once activated, MDEV will continue to replicate in M. dunni cells and can infect many other cell types (4). MDEV is endogenous to M. dunni wild mice (also known as Mus terricolor) at a proviral copy number of one to two per genome, and there is at least one other element in dunni cells that hybridizes to MDEV probes, but it has a different restriction map (4). MDEV has not been found in the genomes of laboratory mice or in mammalian genera other than Mus (4). It is unknown whether MDEV causes pathology or is ever activated in M. dunni mice. MDEV does not interfere with known MLVs, indicating that it uses a different receptor for cell entry (26). MDEV also does not interfere with some nonmurine retroviruses, such as gibbon ape leukemia virus (GALV) (4). The endogenous cat retrovirus RD114 was found to interfere at a low level with MDEV, but this interference was observed in only one cell line (G355 cat glial cells) (4). Furthermore, this interference was weak and varied from one experiment to another, making it unclear whether MDEV and RD114 share a receptor in G355 cells. Other members of the RD114 interference group, such as spleen necrosis virus and Mason-Pfizer monkey virus, have not been found to interfere with MDEV (4). The MDEV receptor is widely expressed among different species, as indicated by the ability of a retroviral vector pseudotyped by MDEV to transduce cells from many species (4). Molecular clones of MDEV were obtained to study its genome and receptor usage (4). However, the clones were unable to produce infectious virus after transfection into permissive cells. Here we describe the correction of a clone that renders it infectious and show that the resulting virus is in the same interference group as the biological isolates. In addition, we have determined the entire sequence of MDEV and describe some unique features of the MDEV genome.