Biomedical subjects

M C Heinrich

Publications and source records attributed to M C Heinrich.

33 records · Page 2Linked to original sources

PubMed1996

Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA.

Animals↗

PubMed1995

Transforming growth factor beta 1 inhibits expression of the gene products for steel factor and its receptor (c-kit).

Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF-beta 1 has been shown to repress human and murine leukemic cell and murine lin- bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF-beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin- hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo.

Acute Disease↗

PubMed1995

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures.

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary long-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-beta 1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified.

Bone Marrow↗

PubMed1994

Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells.

Bone marrow failure is a consistent feature of Fanconi anemia (FA) but it is not known whether the bone marrow failure is a direct and specific result of the inherited mutation or a consequence of accumulated stem cell losses resulting from nonspecific DNA damage. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FACC) plays a regulatory role in hematopoiesis. We exposed normal human lymphocytes, bone marrow cells, endothelial cells, and fibroblasts to an antisense oligodeoxynucleotide (ODN) complementary to bases -4 to +14 of FACC mRNA. The mitomycin C assay demonstrated that the antisense ODN, but not missense or sense ODNs, repressed FACC gene expression in lymphocytes. Treatment with the antisense ODN substantially reduced, in a sequence-specific fashion, cytoplasmic levels of FACC mRNA in bone marrow cells and lymphocytes. Escalating doses of antisense ODN increasingly inhibited clonal growth of erythroid and granulocyte-macrophage progenitor cells but did not inhibit growth of fibroblasts or endothelial cells. The antisense ODN did not inhibit growth factor gene expression by low density bone marrow cells or marrow-derived fibroblasts. We conclude that, while the FACC gene product plays a role in defining cellular tolerance to cross-linking agents, it also functions to regulate growth, differentiation, and/or survival of normal hematopoietic progenitor cells.

Base Sequence↗

PubMed1993

Constitutive expression of steel factor gene by human stromal cells.

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Alternative Splicing↗

PubMed1993

Constitutive and induced expression of hematopoietic growth factor genes by fibroblasts from children with Fanconi anemia.

The pathophysiological abnormalities leading to marrow failure and leukemogenesis in children with Fanconi anemia (FA) are not understood. We tested the hypothesis that the Fanconi anemia mutation results in insufficient production of hematopoietic growth factors by stromal cells by quantifying constitutive and induced production of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and steel factor (SF) by untransformed fibroblasts from eight patients with FA from five different families. While no abnormalities were noted in SF or M-CSF production, we noted substantial variability in IL-6, GM-CSF, and G-CSF responses of cells obtained from different FA patients. Responses ranged from blunting to augmentation when compared to normal controls. Because there was variation between fibroblast strains from affected members of two multiplex sibships, however, it is clear that neither augmentation nor blunting is a direct effect of the FA mutations. In addition, because there was discordance between the G-CSF responses and the GM-CSF and IL-6 responses, the abnormalities noted in IL-1 responsiveness must lie distal to IL-1 receptor function and to stimulus-response coupling pathways shared between the three cytokines.

Cells, Cultured↗

PubMed1992

Specific repression of granulocyte-macrophage and granulocyte colony-stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL-1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.

Animals↗

PubMed1992

Magnesium-dependent thermostability of DNase I.

DNase I can be used to remove contaminating DNA from RNA samples. Heat treatment has been recommended as a method to inactivate DNase I enzymatic activity, thereby allowing subsequent reverse transcription and PCR amplification of DNase I treated RNA. We have found that inactivation of DNase I by heat treatment is strongly dependent on Mg2+ concentration. In addition, deoxyribonucleolytic activity of "inactivated" enzyme may be restored by changes in Mg2+ concentration following heat treatment. Caution should be exercised when using heat treatment alone as a method of DNase I inactivation.

Deoxyribonuclease I↗

PubMed1992

A method for rapid analysis of peripheral blood mononuclear leukocyte cytokine mRNA.

Interleukins, hematopoietic growth factors, and adhesion molecules mediate multiple components of the inflammatory response. Conventional methods for purification of cytokine-producing cells are time consuming and can, therefore, depending on the conditions of cell purification and the nature of the mRNA being purified, result in both over- and underestimates of mRNA content. Therefore, it has been difficult to quantify such factors in ways that are unambiguously reflective of in vivo cytokine production. We describe herein a method for such rapid preparation of peripheral blood mononuclear leukocytes (PBML) that amounts of cytokine mRNA derived from these cells, apart from readily quantifiable losses during RNA preparation, will be reflective of quantities in vivo. Small quantities (10(5) of normal PBML were isolated by density centrifugation of heparinized blood for 1.25 min in a capillary tube. Poly(A)+ RNA isolated by oligo(dT)-cellulose column chromatography was sufficient to reverse transcribe both antigen-specific T-cell-receptor beta-chain mRNA and interleukin 6 (IL-6) mRNA and subsequently amplify the cDNA with Taq polymerase in reverse transcription-polymerase chain reactions (RT-PCR). Although IL-6 mRNA was not present in normal PBML, PBML incubated in culture medium for only 3 h contained approximately one molecule per cell. Because of its rapidity this technique will permit quantification of cytokine mRNA in the steady state and in clinical setting of inflammation. Because the method requires only a small quantity of blood it can be applied to clinical research studies involving children.

Base Sequence↗

PubMed1991

Vascular endothelial cells and hematopoiesis: regulation of gene expression in human vascular endothelial cells.

Vascular endothelial cells do not constitutively produce significant quantities of hematopoietic growth factors, interleukins, or adhesion molecules, but they can be induced to do so by a variety of stimuli including the inductive cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha, as well as phorbol esters, bacterial proteins, endotoxin, certain viruses, and modified low-density lipoproteins. Recently, some groups have begun to investigate the molecular mechanism by which expression of these genes is regulated. Because induced expression of the gene products is always prefaced by an increase in mRNA the focus of attention has been largely upon the mechanisms involved in the process of transcript accumulation. Certain inducing agents drive transcription of these genes, others may induce both transcription and transcript stability. In the case of the inducing factor IL-1, it was recently demonstrated that accumulation of G-CSF, GM-CSF, and interleukin-1 beta mRNAs induced in vascular endothelial cells occurs as a result of transcript stabilization. Based on preliminary studies in a cell-free system, it is proposed that the inductive capacity of interleukin-1 results, at least in part, from its capacity to activate cytoplasmic inhibitors of selective ribonucleases and hypothesize that this may be a mechanism that has been conserved throughout evolution. Because other inducing agents also incite IL-1 gene expression, transcript stabilization may be a common ingredient of most inductive stimuli.

Endothelium, Vascular↗

PubMed1990

Identification of a cell-surface DNA receptor and its association with systemic lupus erythematosus.

We have previously reported the existence of a cell-membrane-associated molecule on human PBMC, which binds DNA and has the characteristics of a receptor. Monoclonal antibodies have been made to this receptor and have been used successfully for the purification of this cell-surface molecule. Preliminary studies have indicated a receptor for DNA on murine kidney and spleen cells which is similar in molecular weight to the human DNA receptor (30 kD). The occurrence of autoantibodies to cell-surface receptors has been described in several autoimmune diseases and we have noted that the serum of patients with lupus and similar disorders inhibit the binding of labeled DNA to human leukocytes. Using a "dot-blot" assay with affinity-purified human DNA receptor, sera from patients with various CTD and from healthy volunteers were screened for anti-receptor antibodies; anti-receptor antibodies were found in many patients with CTD and some of their first-degree relatives. The prevalence of anti-receptor antibodies in normal blood donors was less than 2%. It is hypothesized that anti-receptor antibodies represent an early immune response in lupus and kindred disorders and that anti-DNA antibodies may arise from the corresponding anti-idiotypic response.

Antibodies↗

PubMed1990

Interleukin-1 stimulation stabilizes GM-CSF mRNA in human vascular endothelial cells: preliminary studies on the role of the 3' AU rich motif.

To date, the notion that the AU rich motif in the 3' UT of GM-CSF mRNA can function as an IL-1 response element has not been adequately examined. We suspect that it must play some role because virtually all IL-1 inducible gene have AU rich 3' untranslated regions. Thus, we will continue to address these technical problems so that the hypothesis can be more clearly tested. Nonetheless, we can state with reasonable certainty that: 1) IL-1 induces expression of GM-CSF in EC by inducing accumulation of mRNA, 2) the GM-CSF gene is constitutively transcribed but the half life of the mRNA is short, 3) IL-1 induced mRNA accumulation results from stabilization of the transcript, 4) although most IL-1 responsive genes have AU-rich domains in their 3' untranslated regions, it is not yet clear that these AU-rich regions are sufficient to function as an IL-1 response element, and 5) murine L cells cannot be used for studies on the molecular biology of cytokine induction by IL-1.

Animals↗

PubMed1988

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes.

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.

Antibodies, Monoclonal↗

PubMed1985

Metastatic intravagal paraganglioma. Case report and review of the literature.

Intravagal paragangliomas are rare tumors of neural crest origin. These tumors are usually benign with few reports of metastases or aggressive behavior. One case of intravagal paraganglioma metastatic to a regional lymph node is described and the diagnosis and management of these tumors are discussed. The reports of 14 other cases with regional or distal metastases are reviewed.

Adult↗

PubMed1985

Thrombophlebitis and cellulitis due to Campylobacter fetus ssp. fetus. Report of four cases and a review of the literature.

Four cases of acute thrombophlebitis and cellulitis due to C. fetus ssp. fetus are reported, with a review of 18 previously reported cases. Vascular infection with thrombophlebitis due to C. fetus ssp. fetus occurred predominantly in adult male patients with underlying debilitating, immunocompromising illnesses resulting in a mortality rate of 32%. Although approximately one-third of the patients had exposure to known reservoirs of C. fetus ssp. fetus, none of the patients presented with diarrhea, and only one of the cases had C. fetus ssp. fetus recovered from stool culture. Diagnosis of C. fetus ssp. fetus thrombophlebitis or cellulitis is based on clinical suspicion and recovery of the agent from blood culture; the latter requires an average incubation period of 8 days. Empiric therapy with erythromycin, and an aminoglycoside or chloramphenicol is recommended in suspect patients pending results of blood cultures.

Adult↗