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Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication

In this report,we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially,we found that fresh UCB isolated with the CD34brCD38 CD33 phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition,following ex vivo gene transfer,this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38 CD33 cells. Proliferation of the CD34brCD38 CD33 cells was found to be a prerequisite for efficient transduction. However,in all conditions tested,proliferation of the CD34brCD38 CD33 cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression,an increase in CD38/CD33 expression,and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38 CD33 cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report. View Publication

Wine polyphenols were examined for their capacity to protect the lipid and protein moieties of porcine low density lipoproteins (LDL) during oxidation. The efficiency of resveratrol (3,4',5,trihydroxystilbene) and defined flavonoids was compared to that of a wine extract (WE) containing 0.5 g/g proanthocyanidols. The efficiency of resveratrol for protecting polyunsaturated fatty acids (PUFA) was higher than that of flavonoids in copper-induced oxidation and lower in AAPH (radical initiator)-induced oxidation. The LDL receptor activity was evaluated by flow cytometry using LDL labeled with fluorescein isothiocyanate (FITC) and Chinese hamster ovary cells (CHO-K1). The incubation of CHO-K1 with FITC-LDL oxidized for 16 h reduced the proportion of fluorescent cells from 97% to 4%. At a concentration of 40 microM,resveratrol and flavonoids completely restored the uptake of copper-oxidized LDL and AAPH-oxidized LDL respectively. Total fluorescence could also be obtained with 20 mg/L of WE with both oxidation systems. These data are consistent with previous findings relative to the formation of degradative products from PUFA. They confirm that resveratrol was more effective than flavonoids as a chelator of copper and less effective as a free-radical scavenger. Moreover,they show that WE,which contained monomeric and oligomeric forms of flavonoids and phenolic acids,protected LDL by both mechanisms. View Publication

A novel homology model of the kinase domain of Janus kinase (JAK) 3 was used for the structure-based design of dimethoxyquinazoline compounds with potent and specific inhibitory activity against JAK3. The active site of JAK3 in this homology model measures roughly 8 A x 11 A x 20 A,with a volume of approximately 530 A3 available for inhibitor binding. Modeling studies indicated that 4-(phenyl)-amino-6,7-dimethoxyquinazoline (parent compound WHI-258) would likely fit into the catalytic site of JAK3 and that derivatives of this compound that contain an OH group at the 4' position of the phenyl ring would more strongly bind to JAK3 because of added interactions with Asp-967,a key residue in the catalytic site of JAK3. These predictions were consistent with docking studies indicating that compounds containing a 4'-OH group,WHI-P131 [4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline],WHI-P154 [4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline],and WHI-P97 [4-(3',5'-dibromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazolin e],were likely to bind favorably to JAK3,with estimated K(i)s ranging from 0.6 to 2.3 microM. These compounds inhibited JAK3 in immune complex kinase assays in a dose-dependent fashion. In contrast,compounds lacking the 4'-OH group,WHI-P79 [4-(3'-bromophenyl)-amino-6,7-dimethoxyquinazoline],WHI-P111 [4-(3'-bromo-4'-methylphenyl)-amino-6,7-dimethoxyquinazoline],WHI-P112 [4-(2',5'-dibromophenyl)-amino-6,7-dimethoxyquinazoline],WHI-P132 [4-(2'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline],and WHI-P258 [4-(phenyl)-amino-6,7-dimethoxyquinazoline],were predicted to bind less strongly,with estimated K(i)s ranging from 28 to 72 microM. These compounds did not show any significant JAK3 inhibition in kinase assays. Furthermore,the lead dimethoxyquinazoline compound,WHI-P131,which showed potent JAK3-inhibitory activity (IC50 of 78 microM),did not inhibit JAK1 and JAK2,the ZAP/SYK family tyrosine kinase SYK,the TEC family tyrosine kinase BTK,the SRC family tyrosine kinase LYN,or the receptor family tyrosine kinase insulin receptor kinase,even at concentrations as high as 350 microM. WHI-P131 induced apoptosis in JAK3-expressing human leukemia cell lines NALM-6 and LC1;19 but not in melanoma (M24-MET) or squamous carcinoma (SQ20B) cells. Leukemia cells were not killed by dimethoxyquinazoline compounds that were inactive against JAK3. WHI-P131 inhibited the clonogenic growth of JAK3-positive leukemia cell lines DAUDI,RAMOS,LC1;19,NALM-6,MOLT-3,and HL-60 (but not JAK3-negative BT-20 breast cancer,M24-MET melanoma,or SQ20B squamous carcinoma cell lines) in a concentration-dependent fashion. Potent and specific inhibitors of JAK3 such as WHI-P131 may provide the basis for the design of new treatment strategies against acute lymphoblastic leukemia,the most common form of childhood cancer. View Publication

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

OBJECTIVE To investigate the inhibition of matrix metalloproteinase 1 (MMP-1),MMP-8,and MMP-13 by doxycycline,and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS Recombinant human MMP-1 (collagenase 1),MMP-8 (collagenase 2),and MMP-13 (collagenase 3),truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain,and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen,a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline,while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast,in experiments with the peptolide substrate,neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline,while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8,inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8. View Publication

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7,termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity,some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity,several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus,as well as sequence following the Sec7 domain,also had high activity. The mutant lacking 630 N-terminal amino acids was,however,only 1% as active,as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5,and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated,but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at textless/=50 microM. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus,the Sec7 domain of p200,like that of Sec7 itself (Sata,M.,Donaldson,J. G.,Moss,J.,and Vaughan,M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,4204-4208),plays a role in BFA inhibition as well as in GEP activity,although the latter is markedly modified by other structural elements. View Publication

Analysis of the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen data using the COMPARE algorithm to detect similarities in the pattern of compound action to flavopiridol,a known inhibitor of cyclin-dependent kinases (CDKs),has suggested several possible novel CDK inhibitors. 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one,NSC-664704 (kenpaullone),is reported here to be a potent inhibitor of CDK1/cyclin B (IC50,0.4 microM). This compound also inhibited CDK2/cyclin A (IC50,0.68 microM),CDK2/cyclin E (IC50,7.5 microM),and CDK5/p25 (IC50,0.85 microM) but had much less effect on other kinases; only c-src (IC50,15 microM),casein kinase 2 (IC50,20 microM),erk 1 (IC50,20 microM),and erk 2 (IC50,9 microM) were inhibited with IC50s less than 35 microM. Kenpaullone acts by competitive inhibition of ATP binding. Molecular modeling indicates that kenpaullone can bind in the ATP binding site of CDK2 with residue contacts similar to those observed in the crystal structures of other CDK2-bound inhibitors. Analogues of kenpaullone,in particular 10-bromopaullone (NSC-672234),also inhibited various protein kinases including CDKs. Cells exposed to kenpaullone and 10-bromopaullone display delayed cell cycle progression. Kenpaullone represents a novel chemotype for compounds that preferentially inhibit CDKs. View Publication

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (approximately 180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP,of course). Cholera toxin was the first to be recognized,hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking,ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms,which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells,it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain,a sequence of approximately 200 amino acids that resembles a region in the yeast Sec7 gene product,which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains,among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity,recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4,5,or 6. View Publication

Fetal liver,the major site of hematopoiesis during embryonic development,acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps,the molecular events accompanying this process are just beginning to be characterized. In this study,we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM),an interleukin-6 family cytokine,in combination with glucocorticoid,induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes,expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations,livers from mice deficient for gp130,an OSM receptor subunit,display defects in maturation of hepatocytes. Interestingly,OSM is expressed in CD45(+) hematopoietic cells in the developing liver,whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells,transiently expanding in the fetal liver,produce OSM to promote development of hepatocytes in vivo. View Publication

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor,these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study,evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation,a non-JAK2-dependent signal,in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates,STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1,-3,-5a,and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT,JAK3/AP-1,and JAK3/MAPK pathways and their cellular consequences. Taken together,these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways. View Publication

Human mesenchymal stem cells are thought to be multipotent cells,which are present in adult marrow,that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues,including bone,cartilage,fat,tendon,muscle,and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic,chondrocytic,or osteocytic lineages. Individual stem cells were identified that,when expanded to colonies,retained their multilineage potential. View Publication