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Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.

Vpr, an accessory gene product of HIV-1, is incorporated into cells when added to the culture medium. Via such function Vpr has been shown to transduce a protein into cells that is expressed as a chimeric protein with Vpr. The domain required for protein transduction, however, remained to be clarified. Here we identified a sequence encompassing 52-78 amino acids of Vpr (C45D18) that enables nuclear trafficking of proteins. When chemically synthesized C45D18 was added to the culture medium of human cord blood mononuclear (CBMN) cells, most cells became positive for the incorporated C45D18. Furthermore, recombinant proteins conjugated with the C45D18 were efficiently transduced and transported to regions corresponding to the nucleus. Incorporation of C45D18-conjugated protein was observed within a few hours after addition of the protein, independent of cellular growth. Although it is well known that Tat-derived peptide has a transducing activity, C45D18 was more active than Tat peptide for trafficking proteins into cells. Taking together with results from FACS analysis revealing that more than 90% of CBMN cells were positive for X-gal staining after treatment of C45D18-conjugated beta-galactosidase, we propose that C45D18 translocates bioactive macromolecules directly into the nucleus.

Vpr, an accessory gene product of HIV-1 which induces cell cycle abnormality leading to the increased HIV replication, is supposed to be a possible target for anti-AIDS drugs. We recently established a cell line (MIT-23) in which Vpr-induced cell cycle perturbation could be manipulated by a tetracycline promoter. Here, we screened anti-Vpr activity in 27 kinds of herb drugs using MIT-23 cells. One of the extracts prepared from Houttuyniae herba showed an inhibitory activity. Quercetin (QCT), a compound of this crude drug, efficiently inhibited Vpr function without affecting its expression. Furthermore, data suggested that Vpr-induced transcription from HIV-LTR was considerably abrogated by QCT. These data indicate that QCT, a flavonoid previously reported to inhibit HIV replication, also targets Vpr, implicating that MIT-23 cell provides a novel strategy for screening compounds possessing anti-Vpr activity which would be in turn utilized for clarifying the mechanism of Vpr function., (Copyright 1999 Academic Press.)

We investigated the cytokine-specific involvement of two members of the microtubule-associated protein kinase family, extracellular signal-regulated kinase (ERK)(1 and 2) and p38, in normal human neutrophils. Both tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced tyrosine phosphorylation of a 42-kDa protein in human neutrophils, though the time course of its phosphorylation and its band pattern in electrophoresis differed for each of the cytokines. In addition, GM-CSF, but not TNF, induced a mobility shift of 42-kDa ERK2 in human neutrophils. By using immunoprecipitation followed by immunoblotting, we clarified that GM-CSF, but not TNF, induced tyrosine phosphorylation of ERK2 and that TNF, but not GM-CSF, induced tyrosine phosphorylation of p38. Results of a combined stimulation study showed that tyrosine phosphorylation of ERK2 and that of p38 do not interfere or interact with each other at least in human neutrophils. These results indicate cytokine specific involvement and an independent activating system of ERK and p38 in normal human neutrophils stimulated by two cytokines which share many biological activities in these cells.

We evaluated the interrelationship between the respiratory activity and amount of proteins responsible for this function in normal and subnormal human phagocytes, neutrophils, eosinophils, monocytes, and macrophages. The superoxide-producing capacity was eosinophils > neutrophils > monocytes = macrophages when the cells were stimulated with chemotactic peptide or phorbol ester. Consonant with this finding, the protein content of three essential components of phagocyte oxidase (p22-phox, p67-phox, and p47-phox) was also eosinophils > neutrophils > monocytes = macrophages. On the other hand, the amount of another essential component, gp91-phox, was macrophage > neutrophils > eosinophils > monocytes. These findings together indicate an overall positive interrelationship between protein content and its responsible function, though only gp91-phox was not associated with the functional capacity and low amounts of this component supported the increased respiratory burst activity of eosinophils.

CD4+ cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1- cells (CD4hiGr-1-) and CD4+ Gr-1+ cells (CD4loGr-1lo). CD4hiGr-1- cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1lo cells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1lo cells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1lo cells were present in the recipients' BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr-1lo cells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.

The p53 gene is currently thought to be a tumor suppressor gene, and its alterations have been suggested to be involved in the pathogenesis of several human malignancies, including some leukemias and lymphomas. We present here evidence for the possible involvement of p53 gene mutations in the myelodysplastic syndrome (MDS), although the incidence is relatively low. Forty-four patients with MDS and six patients with overt leukemias that developed from MDS were studied for p53 gene alterations using reverse transcriptase-polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Three patients with MDS (2 RAEB and 1 RAEB in T) had missense point mutations in the conserved regions of the p53 coding sequence. Furthermore, expression of the wild-type p53 mRNA was not detected in these three patients. The probable absence of normal p53 function in the three cases studied here suggests that alterations in the p53 gene may occasionally play a role in MDS. These three MDS patients with p53 gene mutations and an MDS-derived erythroleukemia cell line that we had previously reported to carry a p53 gene mutation showed no N-ras gene mutations, suggesting heterogeneity in the oncogenic mechanism of MDS.

The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor-dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.

The effects of hematopoietic growth factors on human monocyte superoxide (O2-) release were investigated by using purified human monocytes in suspension. Among growth factors studied, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), and interleukin-3 (IL-3) primed human monocytes and enhanced O2- release stimulated by the receptor-mediated agonists, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate, which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of cells with 1 to 5 ng/mL (0.07 to 0.34 nmol/L) GM-CSF, 50 to 100 ng/mL (0.5 to 1.1 nmol/L) M-CSF, or 10 to 20 ng/mL (0.6 to 1.3 nmol/L) IL-3 for 10 minutes at 37 degrees C. Potency of the maximal priming effects on FMLP- or Con A-induced O2- release was GM-CSF greater than M-CSF = IL-3. The combination of the optimal concentrations of any two CSFs resulted in the effect of more potent priming agent alone. Enhancement of O2- release by GM-CSF was observed over the complete range of effective concentrations of FMLP (10(-8) to 10(-6) mol/L). The pretreatment of monocytes with granulocyte-CSF (50 ng/mL), interferon-gamma (1,000 U/mL), or IL-4 (20 ng/mL) for 10 minutes at 37 degrees C had no effect on O2- release stimulated by FMLP or Con A. These findings show that GM-CSF, M-CSF, and IL-3 selectively enhance O2- release in human monocytes triggered by receptor-mediated agonists after short-term preincubation.

Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time-courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM-CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

We have established a new nonlymphoid leukemic cell ine from a patient with myelodysplastic syndrome (MDS), which progressed to overt leukemia. The parental cell line and a subline derived from this line have absolute dependency on several cytokines for their long-term survival and growth. The parental line designated F-36P requires granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for continuous growth, while a subline designated F-36E can be maintained in the presence of erythropoietin (Epo) alone. When these cytokines are depleted, both the parental and the subline cells die within several days, even in medium supplemented with fetal calf serum (FCS). F-36E, maintained in the presence of Epo, constitutively synthesizes hemoglobin at a significant level. F-36P, which is usually maintained in the presence of GM-CSF or IL-3, can be induced to synthesize hemoglobin when GM-CSF or IL-3 is substituted by Epo. The surface marker profile shows that the F-36P cells are positive for the leukocyte common antigen (CD45) and some common multilineage markers such as CD13, CD33, and CD34, and negative for T- and B-cell antigens and mature myelomonocytic antigens. However, some monoclonal antibodies recognizing erythroid and platelet glycoproteins react with these cells. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in a multipotent stem cell. It is also evident that the F-36 cells can be induced to differentiate into the erythroid lineage in the presence of Epo. This, to our knowledge, is the first description of a human leukemic cell line that can be stimulated to synthesize hemoglobin by Epo.

Granulocyte colony-stimulating factor (G-CSF) was reported to increase the number of colony-forming units-spleen (CFU-S) and multilineage colonies as well as myeloid-committed cells. We investigated the effects of G-CSF on myeloid progenitors and primitive stem cells in a mouse bone marrow transplantation (BMT) system. Lethally irradiated mice received BM cells from untreated or 5-fluorouracil-treated mice, and then were administered G-CSF or carrier buffer (control) for 5 days from immediately after BMT. A pre-CFU-S assay was performed by the repeated transplantation of BM cells from the first BMT recipients to other mice. By the method of polymerase chain reaction, most of the spleen colonies in the secondary recipients were confirmed to be derived from the first donors. G-CSF did not increase the peripheral white blood cell count significantly, but did increase the number of immature myeloid cells and granulocyte-macrophage colony-forming cells in the BM. The number of erythroid cells in the BM was initially suppressed and then increased by G-CSF treatment. In addition, the pre-CFU-S assay showed an increase in pre-CFU-S cells due to G-CSF administration. The number of spleen colonies of first BMT recipients did not increase, but a higher percentage of them were committed to a certain lineage by G-CSF treatment. These findings suggest that G-CSF has important roles in the early stages of hematopoiesis.

We characterized the molecular species of human macrophage colony-stimulating factor (hM-CSF) found in serum and urine, using immunoblot analysis after partial purification on an antibody-bound affinity column. Although antibodies were prepared using the recombinant product of the large form of hM-CSF with a molecular weight (MW) of 85 Kd as the antigen, this immunoblot system was also capable of detecting the small form of hM-CSF with a MW of 40 to 60 Kd. A single band with a MW of 43 Kd, which reacted with anti-recombinant hM-CSF IgG but not with control IgG, was found when serum and urine from normal adults underwent electrophoresis on reduced sodium dodecyl sulfate-polyacrylamide gel and subsequent immunoblotting. This band represented a subunit of the large form of hM-CSF, because the large form of hM-CSF is a homodimer of a subunit with a MW of 43 Kd and the small form of hM-CSF is a homodimer of a subunit with a MW of 20 to 30 Kd. Analysis of serum and urine from leukemic patients and pregnant women, who had higher serum levels of hM-CSF than normal adults, showed only a single band with a MW of 43 Kd as a hM-CSF-specific molecule. These results suggest that the large form of hM-CSF is the major species in human body fluids.

p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase-polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenström's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias.

A recently discovered immunosuppressive agent, FK506, has been shown to be effective primarily as an inhibitor of T cell responses in vitro, but little is known about its effects on accessory cell function. This study was undertaken to determine the effect of FK506 on interleukin 1 (IL-1) production by macrophages, by using a sensitive and specific enzyme-linked immunosorbent assay. FK506 partially suppressed IL-1 alpha release, from macrophage-like U937 cells stimulated with phorbol myristate acetate and from human monocytes and alveolar macrophages activated with lipopolysaccharide, in a dose-dependent manner. Moreover, it was indicated that FK506 suppressed not only IL-1 release but also IL-1 synthesis itself, by measurement of cell-associated IL-1 alpha of U937 cells. The optimal concentrations of FK506 for suppressing IL-1 alpha did not affect cell viability or proliferation, and were 10- to 100-fold lower than those of cyclosporin A. It is concluded that FK506 affects macrophage physiology, suppressing IL-1 alpha production significantly. Thus, FK506 has the potency to act on non-T cells and the effect on macrophages may play an additional role in preventing graft rejection.

A recombinant hst-1 protein produced in silkworm cells by a recombinant baculovirus, previously shown to be a potent mitogen for NIH3T3 cells and human endothelial cells, also stimulated anchorage-independent growth of NRK-49F cells. Amino acid sequence analysis revealed that the amino-terminal sequence with 58 amino acids was cleaved off in silkworm cells. These results indicated that the mature hst-1 protein consisting of 148 amino acids had transforming growth factor activity.

We have investigated the effects of recombinant human tumor necrosis factor (TNF)/cachectin on the cellular binding of human low density lipoprotein (LDL) to human skin fibroblasts. When recombinant TNF was added to cultured cells, LDL binding doubled after 24 h of incubation. The effect of TNF was dose-dependent and its maximal effect was observed at concentrations of 1-10 ng/ml. TNF also stimulated the growth of human skin fibroblasts 1.6-fold. These results indicate that TNF increases LDL-receptor activity, which might be related to its stimulatory effect on cell growth.

We investigated the clinical value of measuring serum concentrations of soluble IL-2R in monitoring sarcoidosis. Serum concentrations of soluble IL-2R were measured in 70 patients with sarcoidosis. The mean value for active untreated sarcoidosis was 1,143 +/- 509 U/ml, while the normal range in 97 healthy control subjects was 80 to 300 U/ml. The mean value for active untreated sarcoidosis was significantly higher than that for dormant disease (353 +/- 183 U/ml) or that for corticosteroid-treated patients (380 +/- 151 U/ml). Serial changes in serum soluble IL-2R level were studied in cases of spontaneous remission or in corticosteroid-treated patients; a good correlation was noted between the changes in serum level of soluble IL-2R and clinical status. A positive correlation was noted between serum concentration of soluble IL-2R and serum ACE activity. These data confirmed that measurement of serum concentration of soluble IL-2R could be used in monitoring the disease activity in sarcoidosis.

Granulocyte colony-stimulating factor(G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increased neutrophil C3bi-receptor expression and adherence and rapidly (less than 10 min) primed neutrophils to enhanced O2- release and membrane depolarization stimulated by chemotactic peptide. Direct triggering of O2- release in suspended neutrophils was also provoked by GM-CSF but not by G-CSF. GM-CSF-induced O2- release was inhibited by cyclic AMP agonists and cytochalasin B. The biological activity was greater in non-glycosylated GM-CSF than in glycosylated GM-CSF, whereas it was identical in glycosylated and non-glycosylated G-CSFs. Direct stimulation and priming by GM-CSF were consistently greater than those by G-CSF and the combined addition of the optimal concentrations of G-CSF and GM-CSF resulted in the effects of GM-CSF alone. These findings indicate that the effects of G-CSF and GM-CSF on neutrophil functions are qualitatively and quantitatively different from each other.

Insulin gene expression is restricted to pancreatic B cells and the 5' flanking region is responsible for the tissue specificity. The GCCATCTG motif in this region of the rat insulin 1 gene functions as an enhancer for insulin transcription. A cDNA coding for a GCCATCTG motif-binding protein (IEBP1) was isolated from a rat pancreatic B cell tumor lambda gt11 library. The IEBP1 protein was found to be the rat counterpart of the immunoglobulin (Ig) enhancer binding protein E12/47 having a helix-loop-helix domain. This result indicates that the Ig gene and insulin gene employ the same (or a similar) binding protein as a part of their transcriptional apparatus.

Recombinant latent form of transforming growth factor-beta 1 (L-TGF-beta 1) is activated by various chemical treatments, including acidification and heating. However, cellular mechanisms that release transforming growth factor-beta (TGF-beta) in an active form have not been fully elucidated. Investigated herein are the effects of L-TGF-beta 1 on various leukemic cell lines. Heat-activated L-TGF-beta 1 inhibited colony formation of U937, KG-1 and HL-60, whereas untreated L-TGF-beta 1 had only a marginal effect on these cells. In contrast, colony formation of human erythroleukemia cell line (HEL) was markedly inhibited by both heat-activated and untreated L-TGF-beta 1. In vitro incubation of L-TGF-beta 1 with HEL cells did not release the active form in the culture supernatants. These results suggest that HEL cells are capable of activating L-TGF-beta 1, but only in a cell-associated manner. Since HEL cells produce L-TGF-beta 1, it may act as an autocrine negative growth factor on these cells.

B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus-induced transient aplastic crisis.

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.

Cardiac myosin light chains are released from the infarcted myocardium soon after the onset of infarction, and the level of myosin light chains in the serum reflects infarct size, regardless of the presence of early coronary reperfusion. In this study, we used serum myosin light chains and assessed changes in left ventricular function to evaluate the effects of intracoronary thrombolysis on infarct size, with special reference to the interval between the onset of infarction and the time of reperfusion. Forty patients with acute myocardial infarction who underwent coronary angiography and intracoronary thrombolysis for the left anterior descending artery (LAD) early after the onset of infarction were categorized in four groups: in group A (n = 9) the LAD was already patent at the start of intracoronary thrombolysis, in group B (n = 12) antegrade flow in the LAD was achieved within three hours of the onset of symptoms, in group C (n = 10) antegrade flow was achieved later than three hours after the onset of symptoms, and in group D (n = 9) antegrade flow in the LAD was not achieved. The peak appearance time of creatine phosphokinase (CPK) in group D was significantly later than in the other groups. The total release of CPK in group C (3,119 +/- 414 IU/l) was greater than that in group A (1,332 +/- 346 IU/l) (p less than 0.01), but the total CPK release in group D (2,525 +/- 525 IU/l) was not statistically different from those of any other group. In all four groups myosin light chain I appeared in the serum from the first day on and reached their peak values between the third and fifth days. The peak values of myosin light chain I were 14.7 +/- 2.1 ng/ml for group A, 16.3 +/- 2.3 ng/ml for group B, 24.6 +/- 2.4 ng/ml for group C, and 25.1 +/- 1.8 ng/ml for group D. The peak myosin light chain levels in groups A and B were less than those in groups C and D. Twenty-nine of the 40 patients had no previous episodes of infarction, and these 29 patients were classified in two groups according to the Forrester's subset class on the first day: four of seven patients in group D were in subsets II, III or IV; whereas, the majority of patients in groups A, B and C were in subset I.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein hormone that specifically stimulates both production and functional activation of neutrophils, while interferon-alpha (IFN-alpha) is known to suppress myelopoiesis, including neutrophil production in vivo and in vitro. On a possibility that IFN-alpha may operate as one of the inhibitory feedback factors in neutropoiesis, we examined whether neutrophils produce IFN-alpha in response to G-CSF. Northern blot analysis showed that messenger RNA (mRNA) for human IFN-alpha 1 became detectable time-dependently in highly purified human neutrophils incubated with purified recombinant human G-CSF (rhG-CSF). But such transcription was not observed either in neutrophils incubated with other neutrophil activators, such as formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharides (LPS), or in blood mononuclear cells incubated with rhG-CSF. In addition, radioimmunoassay for human IFN-alpha showed that its levels in culture medium of the rhG-CSF-treated neutrophils rose markedly (up to approximately 100 IU/mL/1 x 10(7) cells) in a time-dependent way, compared with those of nonstimulated neutrophils. These findings suggest that the G-CSF/IFN-alpha system may participate in the feedback regulatory loop of neutropoiesis.

The tritium release assay for the demonstration of thymidylate synthetase activity has been applied to the measurement of enzyme activity in the bone marrow of four patients with pernicious anemia and nine normal subjects. On the average, an approximately ninefold increase in enzyme activity was observed in patients with pernicious anemia. In the absence of 5, 10-methylene-tetrahydrofolate, enzyme activity was reduced in both normal and in pernicious anemia cells. Addition of 5, 10-methylene-tetrahydrofolate to the assay medium resulted in a far greater activation of thymidylate synthetase activity in megaloblastic bone marrow cells than in the cells of control subjects.

We examined whether chemical agents reported to induce differentiation of leukemic cells also have differentiating effects on normal human granulocytes using alkaline phosphatase activity as a marker. Among 11 compounds examined, only vitamin A analogues were shown to induce this activity in granulocytes from bone marrow of normal individuals. Retinoic acid was the most potent inducer of the activity followed by retinal, whereas retinol and retinol acetate did not induce any activity. The effect on the alkaline phosphatase activity by retinoic acid and retinal was considered to reflect their effect on normal granulocytic differentiation and maturation.

The relationship between chromosomal abnormality and oncogene activation was investigated during leukemic progression in two patients with myelodysplastic syndrome (MDS). Both patients had partial or complete deletion of chromosome 5 in metaphase cells obtained throughout the progression to leukemia. Analysis with specific oligonucleotide probes revealed that bone marrow cells containing an activated N-ras oncogene proliferated in a dominant manner during the process of leukemic conversion in both patients. These observations suggest that the chromosomal abnormality may precede activation of the N-ras gene in these patients, and that both the chromosomal abnormality and the activated N-ras oncogene contribute to the development of leukemia.

DNAs from nine out of ten acute leukemia cases that were negative by in vitro focus forming assays exhibited transforming activity tested by in vivo selection assays in nude mice using transfected NIH3T3 cells. Of the nine cases, six cases contained activated N-ras genes, and one case exhibited activation of the c-K-ras gene. None of the ras gene family showed homology with the transforming genes derived from the other two cases. Our observations indicate that in vivo selection assays detect transforming genes including ras oncogenes at high frequency, and that activated N-ras genes are frequently detected in human acute leukemias.

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10-methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10-methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

The lipase released by heparin infusion into perfused rat lungs is shown to be identical with lipoprotein lipase on the basis of the following characteristics: 1) it required plasma for full activity; 2) it was largely inhibited by 1 M NaCl; and 3) it bound to a heparin-Sepharose gel and eluted with buffer containing 1.5 M NaCl. Macrophages prepared from rat lungs released a considerable amount of lipoprotein lipase, with characteristics similar to those of the lipoprotein lipase released from heparin-perfused rat lungs. Pulmonary surfactant-producing type II pneumocytes did not cause a significant release of lipoprotein lipase. No apparent lipoprotein lipase activities were detected in the conditioned medium of other lung cells from which macrophages had been selectively removed.

The regulatory mechanism of differentiation of granulocyte and macrophage precursor cells (G/M CFU-C) in bone marrow and spleen obtained from nude mice bearing colony-stimulating factor (CSF) producing tumor (G-mice), which developed marked granulocytosis, was studied. In these mice, granulopoiesis is enhanced in the spleen, but suppressed in bone marrow. Coculture of G-mouse bone marrow cells with splenic cells of control nude mice (C-mice) and of G-mice resulted in 68% and 62%, respectively, more colonies than expected, while coculture of C-mouse bone marrow cells with these two sources of cell fractions resulted in only 2% and 11% more colonies. In the double-layer agar culture system, bone marrow and splenic cells of C- and G-mice produced a maximal number of colonies containing adequate amounts of human urinary CSF in the upper layer when C-mouse bone marrow cells were added to the lower layer, while these four sources of cells produced a moderate or minimal number of colonies when splenic cells of C- and G-mice or G-mouse bone marrow cells were added to the lower layer. Morphological examination of colonies formed in the upper layer revealed that addition of C-mouse bone marrow cells or irradiated G-mouse bone marrow cells to the lower layer resulted in a massive increment in the number of colonies with pure granulocytes and granulocyte and macrophage mixed (G + G/M) colonies formed by G-mouse bone marrow cells in the upper layer. However, the addition of irradiated C-mouse bone marrow cells or G-mouse bone marrow cells before irradiation to the lower layer did not change G + G/M colony formation by G-mouse bone marrow cells in the upper layer. We could reproduce these findings with conditioned media obtained from 3-day liquid cultures of these cell fractions. This suggests that a diffusible factor may be necessary for inhibition of G + G/M colony formation in G-mouse bone marrow cells. The fine mechanism of such inhibition remains to be clarified.

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.

We recently established a new simple enzymatic assay method for measuring total urinary polyamines. To evaluate the clinical usefulness of measuring total urinary polyamines as a tumor marker, we have applied this method to the assay of polyamines in the urine of cancer patients. Elevation above 3 SD of the normal mean was found in 116 of the 181 patients with cancer (stomach 49/72 [68.1%], colon 22/32 [68.8%], lung 16/24 [66.7%], blood 15/27 [55.6%], liver 3/14 [21.4%], gallbladder 4/4 [100%], and esophagus 7/8 [87.5%]). Total urinary polyamine levels were determined before and after surgery in 36 patients with gastrointestinal cancer (stomach 22 and colon 14). Urinary polyamine levels fell to within the normal range after successful surgery in 23 of 30 patients who had showed elevated levels of urinary polyamines before surgery. Our data indicate that the determination of total urinary polyamines by our new assay may be useful as a tumor marker.

Recombinant cDNA clones that code for apolipoprotein B(apoB) were isolated from a rat liver cDNA library, using synthetic oligonucleotide probe derived from the sequence of human apoB cDNA. The nucleotide and deduced amino acid sequences of the rat apoB clone pRB5, 1.2 kb in length, showed 83% and 84% homology to those of human apoB. Northern blot analysis revealed that rat apoB cDNA probe cross-reacts with human and rabbit apoB mRNA sequences and the size of those mRNAs, approximately 15 kb long, were not discernibly different. In addition, apoB mRNA was abundant only in the liver and intestine. Finally, cholesterol feeding to rats for six weeks resulted in a several-fold increase in the level of apoB mRNA in the liver.

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125I-EPO was cross-linked to two receptors forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.

Eight adult patients suffering from leukocytopenia have been injected by droplet intravenous infusions with partially purified human urinary colony-stimulating factor (CSFHU), which stimulated human monocytes to produce colony-stimulating activity (CSA) in vitro. Three of eight patients were injected with low-dose CSFHU and five with high-dose CSFHU. The infusion of high-dose CSFHU led to a slightly earlier rise in absolute neutrophil counts and a higher CSA level in the serum, as compared to noninjected leukocytopenic patients. There was little toxicity associated with it. Human urinary colony-stimulating factor used in the clinical studies did not have any CSA in vitro in the presence or absence of the patients' sera. These data may suggest that the intravenous infusion of CSFHU increased the serum CSA level, probably through the stimulation of CSA-producing cells in vivo.

Human urinary colony-stimulating factor (CSF-HU) has been partially purified using five purification procedures (preparation II) for clinical use. In phase I study, six healthy volunteers were injected with preparation II of CSF-HU by a single intravenous droplet infusion. Two volunteers received 4 X 10(6) units (U), two received 8 X 10(6) U, and the final two received 1.2 X 10(7) U of preparation II of CSF-HU. Five volunteers did not have any symptoms, but one volunteer who received 1.2 X 10(7) U complained of transient, mild chilliness during the infusion. Liver function test, coagulation test, urine analysis and patterns of electrocardiogram did not change after infusions. In phase II study on preparation II of CSF-HU, forty-four patients with urological malignancy were infused with a daily dose of 8 X 10(6) U CSF-HU for 7 days from the next day after the end of the second courses of two consecutive courses of the same chemotherapeutic regimen. The average nadirs of leukocytes and granulocytes of 33 evaluable cases were higher in CSF-HU-infused courses (second courses) than in control courses (first courses), with statistic significances. The average period under 2,000 leukocytes/mm3 and the average period under 500 granulocytes/mm3 were shorter in CSF-HU-infused courses than in control courses. These results might indicate that infusions of preparation II partially protect the patients from granulocytopenia after anticancer chemotherapies.

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4-8+16+, WT31+ phenotype, low or undetectable non-major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4-8-16+, WT31 phenotype, and strong non-MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non-MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8-16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.

A 64-year-old man with non-Hodgkin's lymphoma suffered a complete collapse of the left upper lobe of the lung. Fibreoptic bronchoscopy demonstrated a large number of distinct polypoid tumours of the lymphoma in the trachea and bilateral sub-segmental bronchi. The left upper lobe bronchus was completely occluded by the tumours, which responded to the combination chemotherapy against the lymphoma. This report presents the seventh case of non-Hodgkin's lymphoma with multiple endobronchial tumours.

We measured immunoreactive atrial natriuretic peptide (ANP) levels in cerebrospinal fluid (CSF) collected from patients with various neurologic disorders requiring diagnostic lumbar puncture. ANP was present in all of the CSF samples from 45 patients (1.7 +/- 0.6 pmol/L, mean +/- SD). CSF ANP levels were not related to the underlying central nervous diseases of the patients, to the presence or the absence of consciousness disturbance, or to CSF osmolalities in individual patients. In 35 patients, the mean ANP concentration in CSF corresponded to 27% of that in plasma, and there was no significant correlation between ANP concentrations in each paired sample. When ANP in pooled CSF was extracted by anti-ANP-agarose and analyzed by reverse-phase high performance liquid chromatography (HPLC), multiple peaks of ANP were found. One of the major ANP peaks was identified as ANP-(103-126) on the basis of its retention time on HPLC and the specificity of the antiserum used in the radioimmunoassay; however, none of other peaks coeluted with ANP-(99-126), ANP-(101-126), ANP-(102-126), ANP-(103-125), or ANP-(105-126). We conclude from these results that ANP is present in human CSF that is differently processed from ANP in the cardiac atrium.

Endothelin, administered into the coronary arteries of anesthetized dogs, produced a profound and long-lasting reduction in coronary blood flow with electrocardiographical evidence of myocardial ischemia. Coronary angiography revealed delayed filling of the distal branches and, in some cases, cessation of the blood flow distal to the epicardial portions of coronary arteries. The coronary vasoconstriction induced by endothelin subsided after intracoronary administration of nitroglycerin. Pretreatment with the Ca2+-channel antagonist, nitrendipine, suppressed endothelin-induced vasoconstriction. These findings suggest that endothelin, produced by vascular endothelial cells, may contribute to the pathogenesis of coronary vasospasm.

We have developed a method of introduction of macromolecules into normal human hemopoietic stem cells. The erythrocyte ghosts were loaded with diphtheria toxin fragment A (molecular weight = 22,000 daltons), which exerts cytotoxicity only in the intracellular space. Granulocyte-macrophage colonies of human bone marrow cells incubated with the above ghosts in the presence of Sendai virus decreased in number to about 10% of the control. This means that the cell fusion and the subsequent introduction of the fragment A into granulocyte-macrophage progenitors occurred at a high incidence (about 90%). This method will be useful to study intracellular events during the proliferation and differentiation of hemopoietic stem cells.