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Biomedical subjects

J M Heard

Publications and source records attributed to J M Heard.

At least 19 recordsLinked to original sources
PubMed1992

Activation pathways and human immunodeficiency virus type 1 replication are not altered in CD4+ T cells expressing the nef protein.

While recent studies in Rhesus monkeys have pointed out the importance of an intact nef gene for the development of acquired immunodeficiency syndrome (AIDS), no biological function has been so far unambiguously attributed to its product. Since Nef has been described to possess GTP-binding properties and to down-regulate CD4 cell surface expression, we looked for evidences of Nef interfering with the transduction of activating signals in human CD4+ T cells. We used a murine leukemia retroviral vector to express the HIV-1BRU nef gene in two permanent tumoral T-cell lines (CEM and Jurkat) and in two nonimmortalized, interleukin-2 (IL2)-dependent, T-cell clones. The single copy recombinant provirus integrated in the genome of these cells directed the synthesis of a 27-kD protein with a half-life greater than 5 h. The levels of expression of cell surface molecules involved in T-cell functions (CD4, CD3, CD28, CD29, IL-2 receptor) were not modified in cell populations expressing Nef. In immunocompetent T-cell clones, cell proliferation and lymphokine production in response to activating stimuli (IL-2, alloantigens, phorbol esters, or antibodies directed against CD2, CD3, CD4, CD28) remained unmodified. Moreover, the presence of Nef did not change the kinetics of human immunodeficiency virus (HIV) infection.

3T3 Cells↗
PubMed1992

Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.

3T3 Cells↗
PubMed1992

Intra-arterial calcium infusion for hydrofluoric acid burns.

Between March 3, 1987 and Sept. 8, 1989, intra-arterial calcium infusions were used to treat 28 patients (38 extremities) suffering hydrofluoric acid (HF) exposures that failed to respond to topical treatments. Although 18 of the injuries occurred at work, only 1 patient was using HF in a concentration greater than 12%. Many of these products were available over the counter. Most patients did not wear protective equipment and had no concept of the danger of injury from the product. Onset of symptoms (pain, erythema and edema) was delayed from 1 to 6 h after exposure. Arterial catheters were placed and the patient was begun on a protocol using 10 ml of 10% calcium chloride diluted with 40 ml normal saline and infused over a 4-h period. Each patient was allowed a 4-8 h rest period before assessing the need for additional infusions. Blood calcium, magnesium, phosphorus, PT, and PTT were monitored. Infusions were repeated until there was no tenderness. Using tenderness as an end-point increased the number of infusions compared to previous reports. The mean number of infusions was 4.1 (range, 1-10). Success (complete healing) was 100%. There was a significant rise in serum calcium, but not to dangerous levels (range 9.3-12.8). There was also a significant fall in magnesium, which reached levels requiring magnesium intravenously. There were no significant changes in phosphorus, PT, and PTT.

Adolescent↗
PubMed1991

Retroviral-mediated gene transfer into hepatocytes in vivo.

Stable gene transfer into hepatocytes might be used to compensate for a genetic deficiency affecting liver function or to deliver diffusible factors into the blood stream. In rats, we have combined retroviral-mediated gene transfer with a surgical procedure in which the liver is temporarily excluded from the circulation and infected in vivo. Partial hepatectomy was performed 24-48 hr before perfusion with virus to induce hepatocyte division and facilitate viral integration. A helper-free recombinant retrovirus coding for beta-galactosidase with nuclear localization was used to score cells that expressed the transgene. For at least 3 months after gene transfer, up to 5% of hepatocytes expressed nuclear beta-galactosidase. Whereas in vitro reimplantation of genetically modified hepatocytes has proved to be inefficient in stably transferring genes into the liver, our approach provides a feasible alternative.

Animals↗
PubMed1991

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.

Amino Acid Sequence↗
PubMed1991

Gene transfer to somatic tissues using retroviral vectors.

Retroviral vectors have successfully been used for stable gene transfer in a number of in vivo situation. Before this approach can reallistically be applied in the treatment of human genetic diseases, the correction of pathological symptoms have to be documented in an animal model. In this perspective, we have chosen to use a strain of mice carrying the gusmps mutation, where homozygotes are deficient for beta-glucuronidase and develop a mucopolysaccharidosis. Two methods which result in the stable introduction of the human beta-glucuronidase cDNA into skin fibroblasts or hematopoietic stem cells are presented. A third method, whereby genes can be stably transferred to the liver is also discussed.

Animals↗
PubMed1990

A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors.

The myeloproliferative leukemia virus (MPLV) is an acute leukemogenic murine replication-defective retrovirus. By sequencing the envelope gene of a biologically active MPLV clone, we found that this region comprises a novel oncogene named v-mpl in phase with two parts of the Friend murine leukemia virus envelope gene. The MPLV env region could encode an env-mpl fusion polypeptide that presents the characteristics of a transmembrane protein. We show that in vitro infection of bone marrow cells with helper-free MPLV readily yields immortalized factor-independent hematopoietic cell lines of different lineages. In mice, the c-mpl proto-oncogene is expressed in hematopoietic tissues as a 3 kb mRNA. Since v-mpl shares strong structural analogies with the hematopoietin receptor superfamily, it is likely that MPLV has transduced a truncated form of an as yet unidentified hematopoietic growth factor receptor.

Amino Acid Sequence↗
PubMed1987

Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene.

Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by "unmarked" progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.

Animals↗
PubMed1987

Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens.

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.

Acetyltransferases↗
PubMed1987

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Acetyltransferases↗
PubMed1986

Long-term culture of bone marrow-derived preleukemic cells from F-MuLV-infected mice.

The replication-competent Friend leukemia virus (F-MuLV) induces leukemias involving three hematopoietic lineages after a latent period of several months. In an attempt to elucidate the early events of the leukemogenic process, we looked for a method allowing the isolation and the long term in vitro maintenance of preleukemic cells. When established as long-term cultures according to the technique described by Dexter et al, bone marrow cells obtained from 7/7 apparently healthy F-MuLV-infected preleukemic mice led to the accumulation of immature myeloblastic cells, and to the generation of permanent myeloblastic cell lines, which in most cases further became tumorigenic in preirradiated recipient animals. The delays required to obtain cell lines were shorter when the duration of the in vivo infection was longer, suggesting that these cells were committed into the leukemogenic pathway before their transfer into culture flasks. The myelomonocytic preleukemic cells exhibited normal sensitivity to purified preparations of CSFs, but acquired the capacity to grow in the absence of exogenous CSF stimulation. Examination of integrated provirus copies demonstrated that the preleukemic cell proliferation involved a single or a few clones which may progress in vitro from a preleukemic to a fully malignant stage without major modifications of the integrated provirus copies.

Animals↗
PubMed1985

The in vitro autocrine secretion of CSFs alone does not account for the longterm growth of murine myeloid leukemic cells in suspension cultures.

To investigate the mechanisms supporting the in vitro longterm growth of murine leukemic myeloblastic cells, factor-dependent and autonomous myeloblastic cells have been examined for their CSF responsiveness, CSF secretion, and autostimulation of growth. Purified CSF-1, GM-CSF, and IL-3 stimulated cloning and proliferation of both autonomous (ACL) and factor-dependent cell lines (FDCL), and were unable to induce differentiation of these cells. Sensitivities to CSFs were similar for ACL, FDCL and normal bone marrow cells. Most of the cell lines secreted CSFs, stimulating colony formation from normal bone marrow cells in bilayer agar cultures. The number of induced granulo-macrophagic colonies was similar in the presence of either ACL or FDCL. The ability to stimulate their own proliferation was similar for both ACL and FDCL in a longterm suspension culture assay as well as in a shortterm cloning assay. These results strongly suggest that the autocrine secretion of CSF-related activities are not sufficient to fully account for the in vitro longterm proliferation of virus-transformed myelomonocytic cells.

Agar↗
PubMed1985

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.

Animals↗
PubMed1985

Ask your pharmacist?

Retail pharmacists should recognize their inadequate knowledge of Ipecac instructions. When confronted with an acute poisoning, these patients should be referred to their physician or nearest regional poison control center (3). Providing improper instructions creates the risks of: Vomiting a substance with which emesis is contraindicated; Unnecessary emesis; and Use of ineffective and unsafe methods of inducing emesis. Only 2 (6.25%) pharmacists gave complete and accurate instructions for the use of Ipecac--with the exception of mentioning the contraindications to emesis. However, both pharmacists referred the caller to their regional poison control center.

Community Pharmacy Services↗
PubMed1984

The expression of the env gene-related gp66 in mouse cells infected with the helper independent Friend leukemia virus is restricted to the myelomonocytic and mastocytic lineages.

The presence of a gp66 closely related to the Friend ecotropic murine leukemia virus gp70 (F-MuLV) has recently been reported (D. Mathieu-Mahul, J. M. Heard, S. Fichelson, S. Gisselbrecht, B. Sola, and C. J. Larsen (1982) virology 119, 59-67). In the present work, characterization of this gp66 was continued. First, immunoprecipitation tests, using cytoplasmic membrane subfractions from one of the myelomonocytic cell lines in which gp66 was first detected, indicated that most of it was associated with rough endoplasmic reticulum. Second, to define the limits of gp66 expression, a variety of hemopoietic cell lines were analyzed for gp66 content. These lines were obtained (a) from various tumors (including erythroleukemias, chloroleukemias, and lymphatic leukemias) induced in susceptible mice by F-MuLV and (b) from long-term bone marrow cultures (LTBMC) infected with F-MuLV. In the latter case, lines of adherent fibroblastoid cells and nonadherent cells with myelomonocytic and mastocytic characteristics were obtained. Although several F-MuLV isolates were used, gp66 was only expressed in myelomonocytic and mastocytic cells. This did not result from in vitro culture conditions as gp66 was also found in fresh cells. These data suggested that a particular processing of the env gene product may exist in both myelomonocytic and mastocytic cell lines. In agreement with this hypothesis, a metabolically unstable gp62 related to MCF gp70 was found in one myelomonocytic cell line expressing MCF virus.

Animals↗
PubMed1984

Multistep virus-induced leukemogenesis in vitro: description of a model specifying three steps within the myeloblastic malignant process.

A helper-independent Friend leukemia virus was used to infect bone marrow cultures. This virus induces myeloblastic leukemia in mice after a long latency period. Infection of the bone marrow cultures resulted in the in vitro production of myeloblastic leukemogenesis after a long latency period. Three steps were observed in the evolution of the infected cultures, and permanent cell lines were derived at each step. This allowed us to individualize three successive events in the course of the myeloblastic transformation: (i) an abnormal responsiveness to the physiological hormone granulo-macrophagic colony-stimulating factor, (ii) the acquisition of growth autonomy, and (iii) the acquisition of in vivo tumorigenicity.

Animals↗