Biomedical subjects

J N Ihle

Publications and source records attributed to J N Ihle.

At least 19 recordsLinked to original sources

PubMed1992

Pharmacokinetics of subcutaneous recombinant human granulocyte colony-stimulating factor in children.

Fifteen children (age 1.2 to 9.4 years) with advanced neuroblastoma were treated with myelosuppressive chemotherapy (cyclophosphamide, cisplatin, doxorubicin) followed by 5 (n = 5), 10 (n = 5), or 15 (n = 5) micrograms/kg recombinant granulocyte colony-stimulating factor (rG-CSF) subcutaneously (SC) once daily for 10 days, starting the day after chemotherapy. Serial serum samples obtained on days 1 and 10 were analyzed for G-CSF activity by a specific proliferation assay using NFS-60 cells. G-CSF serum concentration-time data were best described by a one-compartment model, with zero-order absorption and first-order elimination. After SC injection, absorption was prolonged, with peak concentrations of G-CSF (3 to 117 ng/mL) being reached after 4 to 12 hours. The relatively slow absorption, with a mean elimination half-life of 5.8 hours on day 1 and 4.5 hours on day 10, provided measurable G-CSF concentrations for the entire 24-hour dosing interval in all patients at each dosage level. The median apparent clearance of G-CSF on day 10 was significantly higher than on day 1 (0.57 v 0.31 mL/min/kg, P = .02), and was positively correlated with the absolute neutrophil count (ANC) (r2 = .33, P = .003). Systemic exposure to G-CSF was dose-related, but interpatient pharmacokinetic variability yielded overlap in area under the concentration-time curve (AUC) at all three dosage levels. Stepwise regression analysis showed that G-CSF AUC could be predicted by a model that includes rG-CSF dosage and ANC on the day of administration (r2 = .82, P = .0001).

Absorption↗

PubMed1992

An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer.

Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested "remission" marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

Bone Marrow↗

PubMed1992

Activation of EVI1 gene expression in human acute myelogenous leukemias by translocations spanning 300-400 kilobases on chromosome band 3q26.

Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome 3q26 where the EVI1 gene has been localized. To identify breakpoints, a restriction map that spans 1700 kilobases (kb) of the EVI1 locus was developed by pulsed-field gel electrophoresis. In one case, t(3;3)(q21;q26), a rearrangement was localized to 170-330 kb 5' of the gene. In a second case, t(3;3)(q21;q26), there was a rearrangement 13 kb 5' of the gene. This rearrangement was cloned and shown to be due to the fusion of sequences from 3q21-22 with the EVI1 locus. In the third case, ins(3)-(q21q25q27), there was a rearrangement that mapped 150 kb downstream from the 5' end of the gene.

Base Sequence↗

PubMed1992

Assignment of a novel protein tyrosine phosphatase gene (Hcph) to mouse chromosome 6.

Hematopoietic cell phosphatase (Hcph) was identified by amplification of conserved protein tyrosine phosphatase sequences from a myeloid cell line and is predominantly expressed in hematopoietic cells. Hcph is unique in containing two, tandemly repeated, src-homology 2 domains in the amino terminal region of the phosphatase. Using a genomic probe in interspecific backcross analysis, the murine Hcph gene maps to mouse Chromosome 6 and is tightly linked to the Tnfr-2 and Ly-4 genes.

Animals↗

PubMed1992

Retrovirus-mediated gene transfer as an approach to analyze neuroblastoma relapse after autologous bone marrow transplantation.

Disseminated neuroblastoma is a malignancy of children often treated by intensive chemotherapy/radiotherapy followed by autologous bone marrow transplantation (ABMT). A high proportion of those treated subsequently relapse. It is unknown if relapse is a consequence of residual disease in the patient or of contaminating malignant cells remaining in the infused marrow, which, of necessity, is harvested and stored prior to ablative chemotherapy/radiotherapy. The assumption that residual cells in the infused marrow contribute to relapse has lead to the adoption of marrow purging prior to reinfusion. However, neither the necessity nor the efficacy of the procedure have been established. We now show how retroviral-mediated gene transfer using the LNL6 vector may resolve this issue. Clonogenic neuroblastoma cells in patient marrow can be transduced and the NEOR gene detected by observing individual neuroblastoma cell colony growth in G418, and by polymerase chain reaction (PCR) of individual colonies. Efficiency of transduction is between 0 and 13.5%. If marrow is exposed to LNL6 prior to infusion and marked cells are detected at the time of relapse, this would demonstrate that infused marrow contributed to disease recurrence. The technique could then be used to analyze the efficacy of marrow purging techniques. Since normal progenitor cells from these patients are also marked, the technique can be used to study factors that modify reconstitution and transducibility of infused marrow. Clinical studies using this approach have now begun.

Base Sequence↗

PubMed1992

Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.

Animals↗

PubMed1992

Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.

Amino Acid Sequence↗

PubMed1992

Interleukin-3 and hematopoiesis.

Considerable information has accumulated over the past several years relating to the biological activities of IL-3 which supports that concept that IL-3 has a unique capacity to support the proliferation and differentiation of a wide spectrum of hematopoietic cells. The pleiotropic activity of IL-3 has in turn elicited considerable enthusiasm for clinical trials of IL-3 in humans. Such clinical trials are ongoing and will certainly reveal a variety of conditions that will benefit from IL-3 treatment alone or in combination with other lymphokines. Of particular note are conditions which would benefit from the proliferation and expansion of early myeloid progenitors. These include bone marrow reconstitutions, aplastic anemias, hematopoietic recovery following chemotherapy. A variety of data demonstrate that hematopoiesis is not dependent upon IL-3. Thus, alternative factors must exist that can also support the proliferation and differentiation of hematopoietic stem cells. This observation and the uniquely T cell origin of IL-3 have led to the concept the production of IL-3 has evolved within the immune system as a mechanism by which immunological reactions can broadly affect hematopoiesis. The lack of an essential role in constitutive hematopoiesis would be consistent with the divergence in the structure of the gene that is seen among species. Such a role would also predict that it will be easy to obtain mice in which the gene has been deleted by homologous recombination. Indeed, such mice will be essential to fully define the role that IL-3 has in the development and content of immune responses. Although IL-3 supports the proliferation of a variety of myeloid lineages and cells at various stages of differentiation, relatively little has been uncovered regarding the influence that IL-3 might have no differentiation. The results strongly suggest that IL-3 does not singularly control differentiation. The other extreme would suggest that IL-3 may support proliferation which allows the expression of a genetic program that is determined completely independent of IL-3. The most appealing hypothesis is that IL-3 can influence the outcome of a differentiation program. In particular, it can be hypothesized that a pluripotential stem cell that is proliferating in IL-3 has a somewhat different pattern of differentiation than cells proliferating in the presence of the factors that control constitutive hematopoiesis. Of particular interest are mechanisms by which IL-3 mediates its biological effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Recombinant human TNF-alpha stimulates the secretion of granulocyte colony-stimulating factor in vivo.

Tumor necrosis factor (TNF) is a macrophage-derived cytokine that causes hemorrhagic necrosis of several human tumors in vitro. It has a wide range of biologic effects including stimulation of secretion of both granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) by normal adult lung fibroblasts in culture. No in vivo data are available on the effect of exogenously administered TNF on cytokine production. In the studies reported here, we show that G-CSF accumulates in the serum in vivo in response to recombinant TNF (rTNF) administration. At the peak of the response circulating levels of 2-6 ng/ml of biologically active G-CSF are detectable. Surprisingly, circulating levels of GM-CSF, interleukin-3 as well as a number of other cytokines were not detectable within the limits of the assays. The results indicate that the levels of GM-CSF or interleukin-3 are minimally 100-fold lower than the peak levels of G-CSF. These data illustrate the complex interplay that cytokines have in vivo. Understanding these interactions in humans is crucial to the correct use of this new class of agents in the clinic.

Adenocarcinoma↗

PubMed1991

Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification.

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R-PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Amino Acid Sequence↗

PubMed1991

Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells.

Murine bone marrow cells expressing the cell surface Ag RB6-8C5 were identified by fluorescence-activated cell-sorting analysis using a rat IgG mAb. The fluorescent intensity of RB6-8C5 was variable on bone marrow cells. This made it possible to separate bone marrow cells into distinct subpopulations, RB6-8C5neg, RB6-8C5lo, and RB6-8C5hi cells. Morphologic analysis of the sorted populations demonstrated that the Ag was expressed on myeloid cells. The expression of RB6-8C5 increases with granulocyte maturation, whereas expression is transient on cells in the monocytic lineage. The RB6-8C5hi sorted cells were enriched for end-stage neutrophils (75%), whereas the RB6-8C5lo sorted cells contained more immature myeloid cells and myelocytes (75%). Lymphocytes and macrophages were less than 5% in any RB6-8C5+ population, whereas the erythroid precursors were RB6-8C5neg. The colony forming unit culture (CFU-C) (greater than 90%) were found in the RB6-8C5neg and RB6-8C5lo populations, and all the CFU-granulocyte, erythroid, megakaryocyte, and macrophage (CFU-GEMM) and burst-forming units-erythroid (BFU-E) were in the RB6-8C5neg population. Granulocyte-macrophage-CSFR (GM-CSFR) and IL-1 alpha R were expressed on RB6-8C5hi bone marrow cells, whereas no receptors could be detected on RB6-8C5neg and RB6-8C5lo cells. The expression of the RB6-8C5 Ag can be induced on RB6-8C5neg cells in liquid culture by IL-3 and granulocyte-macrophage CSF. Thus, RB6-8C5 is a myeloid differentiation Ag whose expression can be regulated by cytokines.

Animals↗

PubMed1991

Characterization of three related murine interleukin-3 surface receptor proteins.

Iodinated interleukin-3 (IL-3) can be covalently cross-linked to three specific surface glycoproteins with net molecular masses of 170, 140, and 65-70 kDa under conditions in which ligand internalization and degradation do not occur. These three proteins plus two additional non-ligand-binding proteins of 90 and 55 kDa can be purified by IL-3 affinity chromatography. Comparative two-dimensional analysis of the tryptic digests of these five proteins indicates that the ligand-binding proteins are highly related at the peptide level. Incubation of cells with 125I-IL-3 at 37 degrees C results in rapid time- and energy-dependent internalization and degradation of ligand. Under these conditions only the 140- and 65-70-kDa binding proteins, which can recycle to the surface after internalization, can be identified. The lability of the 170-kDa protein indicates that it may not recycle. Thus, an energy-dependent mechanism is responsible for internalization and may be necessary for any potential interconversion of the higher 170- or 140-kDa proteins to the lower 140- and/or 65-70-kDa binding proteins.

Affinity Labels↗

PubMed1991

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.

Amino Acid Sequence↗

PubMed1991

Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter.

The inappropriate production of the Evi-1 zinc finger protein occurs in retrovirus-induced murine myeloid leukemias and human acute myelogenous leukemias. In murine leukemias, expression of the Evi-1 gene is associated with retroviral insertions either in the Evi-1 locus, which is immediately 5' of the coding region of the gene, or in the genetically linked Cb-1/fim-3 locus. In these studies, we demonstrate by chromosomal walking and pulse field electrophoresis that the Cb-1/fim-3 locus is located 90 kb 5' of the Evi-1 locus. Primary structure analysis of Evi-1 cDNA clones from a Cb-1/fim-3 rearranged cell line (DA-3) demonstrates that transcription initiates 5' of the Evi-1 locus and that the first noncoding exon of the gene is 681 bp larger than previously defined. S1 nuclease protection studies reveal multiple transcription initiation sites within this region. Comparable transcriptional initiation sites were identified in RNA from kidney and ovary, in which the gene is normally expressed, suggesting that retroviral insertions in the Cb-1/fim-3 locus activate transcription from the normal promoter. In one myeloid cell line (DA-3), a single long terminal repeat (LTR) is present in the Cb-1/fim-3 locus. No stable transcripts were detectable from this LTR. In cells with retroviral insertions in the Cb-1/fim-3 locus, one allele of the Evi-1 locus becomes hypermethylated in the 5' region of the gene. Together, these results are most consistent with an LTR-mediated, long-range cis activation of Evi-1 gene expression.

Alleles↗

PubMed1991

Hematopoietic cells express two forms of lyn kinase differing by 21 amino acids in the amino terminus.

cDNAs for the murine lyn protein tyrosine kinase gene were cloned from mouse bone marrow-derived monocytic cells. Comparison of the human and murine genes demonstrated a 94% homology in peptide sequence. Comparable to the human gene, murine lyn was found to be expressed in myeloid and B-lymphoid lineage cells. During the cloning, two types of cDNAs were obtained that differed by the presence (lynA) or absence (lynB) of 63 bp within the amino-terminal coding region of the gene. The genomic structure of the murine lyn gene demonstrates that the two types of lyn transcripts are derived from alternative splicing utilizing an internal splice donor site. Transcripts for both forms were found to be expressed in myeloid cells. lyn-specific antisera detected comparable levels of proteins of 56 and 53 kDa in hematopoietic cells. these 56- and 53-kDa proteins comigrated with proteins produced by in vitro translation or in vivo expression of the lynA and lynB cDNAs, respectively. The two forms had comparable in vitro kinase activities in immunoprecipitates and showed similar peptide patterns, with partial V8 digestion of the in vitro-phosphorylated proteins. The potential significance of the two lyn proteins is discussed.

Amino Acid Sequence↗

PubMed1991

His-1 and His-2: identification and chromosomal mapping of two commonly rearranged sites of viral integration in a myeloid leukemia.

To identify genes that contribute to myeloid leukemogenesis we have cloned viral integration sites from a CasBrM-MuLV-induced interleukin 3-independent myeloid leukemia cell line. Genomic probes derived from cellular sequences flanking two integrated proviruses were used to screen restriction digests of DNAs from a panel of 52 hematopoietic cell lines, 30 of which were established from CasBrM-MuLV- or MoMuLV-induced mouse leukemias. Probes from one integration site (His-1) defined a region that was rearranged in 3/52 cell lines, and probes from a second integration site (His-2) identified a rearrangement in 2/52 cell lines. Both cases of His-2 rearrangements occurred in concert with viral insertions in the His-1 locus. Genetic mapping of these loci using interspecific backcross analysis assigned the His-1 locus to mouse chromosome 2 and the His-2 locus to mouse chromosome 19. In situ hybridization with a probe from the human homologous region mapped the His-1 locus to human chromosome 2q14-q21. No recombinants were observed between His-2 and Gin-1, a common site of provirus integration in Gross passage A MuLV-induced T-cell leukemias, in 131 backcross animals, suggesting that these loci are tightly linked. The His-1 locus maps to mouse chromosome 2 distinct from any known oncogene or common site of integration but near the proximal breakpoint for a deletion that is observed in over 90% of radiation-induced leukemias.

Animals↗

PubMed1991

Granulocyte colony-stimulating factor corrects the neutropenia associated with glycogen storage disease type Ib.

A young woman with glycogen storage disease, type Ib, and chronic neutropenia had severe recurrent infections. In a life-threatening situation, treatment with granulocyte colony-stimulating factor (G-CSF) resulted in the prompt correction of neutropenia. Subsequently, daily G-CSF therapy has allowed the maintenance of a normal neutrophil count and marked clinical improvement over a period of 18 months. The spectrum of neutropenic conditions which are responsive to G-CSF should include this inherited metabolic disorder.

Adult↗

PubMed1990

Phenotypes and mechanisms in the transformation of hematopoietic cells.

Interleukin 3 (IL-3) is a growth factor that supports the proliferation of early hematopoietic stem cells, as well as cells that are committed to a variety of the myeloid lineages. The mechanisms by which IL-3 functions have been studied through the use of a series of IL-3-dependent cell lines isolated from myeloid leukemias or long-term bone marrow cultures. A variety of studies have implicated tyrosine phosphorylation in IL-3 signal transduction. One of the substrates of phosphorylation is a 140 kDa, IL-3-binding protein that is speculated to be the biologically relevant IL-3 receptor. IL-3, through tyrosine phosphorylation, supports viability and growth through the regulation of transcription of a series of genes including c-myc and c-pim-1. The c-myc gene contributes to viability, in part, by regulating the transcription of the ornithine decarboxylase gene. The role of growth factors in differentiation is less clear. By studying IL-3-dependent myeloid leukemia cell lines, two genes have been identified whose altered expression is associated with blocking the ability of the cells to differentiate. The c-myb gene is a nuclear DNA binding protein that has been implicated in myeloid transformation in a number of systems. The Evi-1 gene is a novel gene of the zinc finger family of transcriptional activators. Possible mechanisms by which these genes interfere with normal differentiation are discussed.

Animals↗