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Biomedical subjects

M K Collins

Publications and source records attributed to M K Collins.

At least 73 records · Page 4Linked to original sources
PubMed1992

Superoxide production by normal and chronic granulomatous disease (CGD) patient-derived EBV-transformed B cell lines measured by chemiluminescence-based assays.

We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.

Acridines↗
PubMed1992

Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents.

Murine bone marrow-derived cells, dependent on interleukin 3 (IL-3) for their growth in culture, undergo programmed cell, or apoptosis, upon cytokine withdrawal. Here it is reported that a variety of DNA damaging agents cause a more rapid onset of apoptosis in a factor-dependent cell line, BAF3, deprived of IL-3. In contrast, when cultured in the presence of IL-3, or other growth promoting factors, BAF3 cells are highly resistant to X-irradiation and the cytotoxic drugs etoposide and cisplatin. Overexpression of the bcl2 gene product also protects BAF3 cells from DNA damage. The presence of IL-3 is not required during the initial events of DNA damage or its repair. In the absence of IL-3, cells still complete the repair of DNA breaks within 15 min, and continue to cycle for 5 h. At this time, IL-3 is necessary to prevent the accelerated onset of DNA cleavage from a G2 arrest point.

Animals↗
PubMed1992

Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a 'sub-G1' peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

Animals↗
PubMed1992

Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells.

The death of hemopoietic cells on withdrawal of CSF occurs by a mechanism known as apoptosis characterized by the early degradation of chromatin into oligonucleosome-length fragments. Insulin-like growth factor I plays a pivotal role in the regulation of somatic cell growth as a mediator of growth hormone action. Animals with low levels of circulating IGF-I are more vulnerable to infections and have diminished immune responses. To analyze the possibility of a regulatory role of IGF-I on hemopoiesis and determine its mechanism of action, we have studied the effect of this growth factor on the survival and proliferation of two IL-3-dependent hemopoietic cell lines and in IL-3-responsive primary cultures of bone marrow-derived mast cells. In IL-3-depleted cultures, IGF-I prevented DNA fragmentation and apoptotic cell death. Insulin at high concentration had a weak protective action and IGF-II was inactive in suppressing apoptosis in these IL-3-dependent hemopoietic cells. Cell proliferation was also stimulated by IGF-I in the absence of other hemopoietic growth factors although it was a weak mitogen when compared with IL-3. These results indicate that circulating or locally produced IGF-I may promote survival of both the steady state hemopoietic precursor population and cytokine-producing cells and could therefore regulate hemopoiesis acting in a concerted manner with other CSF.

Animals↗
PubMed1992

Immunity to B16 melanoma in mice immunized with IL-2-secreting allogeneic mouse fibroblasts expressing melanoma-associated antigens.

Co-presentation of weak tumour-associated antigens along with strongly immunogenic determinants leads to the development of an anti-tumour immune response in recipients syngeneic with the tumour. Tumour immunity develops in mice immunized with tumour cells modified by the introduction of cDNA for interleukin-2 (IL-2). Here, we report the anti-tumour response following immunization with an IL-2-secreting cell construct that expresses tumour-associated antigens, along with allogeneic major histocompatibility antigens. The construct was prepared by transfecting LM(TK-) mouse fibroblasts (H-2k) with genomic DNA from B16 melanoma cells syngeneic in C57BL/6J mice (H-2b). Transfectants expressing melanoma-associated antigens (MAA) were then infected with an expression-competent retroviral vector containing a cDNA specifying human IL-2. Cytotoxicity toward B16 cells was detected for as long as 5 months in both spleen and macrophage cell populations in C57BL/6J mice immunized with the IL-2-secreting cells. Mice immunized with non-IL-2-secreting, MAA-positive allogeneic cells developed melanoma immunity as well, but to a lesser extent. Immunity to 2 tumour-cell lines expressing the H-2d haplotype and to YAC-1 cells was detected in peritoneal macrophages, but not in spleen cells from C57BL/6J mice immunized with the cell construct, indicating that the response to B16 cells was only partially specific. C57BL/6J mice immunized with the IL-2-secreting cell construct survived significantly longer, following an injection of viable B16 cells, than mice in various control groups. The contribution of allogeneic antigens to the melanoma immunity was indicated by the failure of mice syngeneic with LM(TK-) cells to develop melanoma immunity following immunization with non-IL-2-secreting, MAA-positive cell constructs. The formation of IL-2 partially compensated for the lack of allogeneic antigens.

Animals↗
PubMed1992

Inhibition of apoptosis by calcium ionophores in IL-3-dependent bone marrow cells is dependent upon production of IL-4+.

Calcium ionophores inhibit apoptosis in the IL-3-dependent cell line BAF3 and maintain the cells in a viable noncycling state. In this report, an identical effect of ionophore was also demonstrated on the multipotent IL-3-dependent progenitor cell line FDCP-MIX and on the primary IL-3-dependent cell population that could be cultured from murine bone marrow. Inhibition of apoptosis required extracellular calcium and could be blocked by cyclosporin A. Nuclei from IL-3-dependent cells were found to lack a calcium-activatable nuclease that degrades chromatin in the linker region between nucleosomes, unlike the nuclei of lymphoid cells. The mechanism of action of calcium ionophore could be divided into two distinct steps. First, ionophore induced the production of a survival factor that stimulated DNA synthesis and was identified as IL-4. Second, ionophore inhibited the cell cycle of the various IL-3-dependent cells. IL-4 production could be inhibited by cyclosporin A and required extracellular calcium, whereas cell cycle arrest did not. This implied that factor production was the step that was necessary for inhibition of apoptosis and maintenance of cell viability. This was confirmed by the use of an anti-IL-4R antibody, which blocked the inhibition of apoptosis induced by calcium ionophores.

Animals↗
PubMed1992

Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27.

The herpes simplex virus (HSV) immediate-early protein ICP27 has been postulated to play an auxillary role in HSV. gene expression, augmenting or inhibiting the activation of different viral promoters by the other immediate-early proteins ICPO and ICP4. Here we show that ICP27 alone can up-regulate gene expression of a retroviral vector containing Moloney murine leukemia virus (MoMuLV) regulatory sequences. This is the first report of an effect of ICP27 on gene expression driven by heterologous virus regulatory sequences. The effect does not involve the region of ICP27 which inhibits gene activation of HSV early gene promoters by ICPO and ICP4, but rather is dependent on the same region of ICP27 as is required to augment the activation of HSV late gene promoters by ICPO and ICP4. This indicates that the two activation effects are likely to operate via the same mechanism. Activation by ICP27 is dependent on the 3' LTR and adjacent region of MoMuLV but is independent of the promoter in the 5' LTR which can be replaced by a heterologous promoter such as that of SV40 without affecting activation by ICP27. The significance of this effect for an understanding of the mechanism of action of ICP27 and its role in regulating the gene expression of HSV and potentially of other viruses is discussed.

3T3 Cells↗
PubMed1992

Retroviral pseudotypes produced by rescue of a Moloney murine leukemia virus vector by C-type, but not D-type, retroviruses.

Human HOS cells containing a Moloney murine leukemia virus (Mo-MLV) recombinant genome were infected by a panel of retroviruses. The C-type viruses simian sarcoma associated virus, feline leukemia virus subgroup B, and the feline endogenous virus RD114 were able to form pseudotypes with the Mo-MLV genome, which transferred a selectable marker gene to target cells; however, Human T cell leukemia virus-1 and the D-type viruses Mason-Pfizer monkey virus and simian retrovirus-1 failed to rescue the Mo-MLV vector. Further characterization of the RD114 pseudotype demonstrated that it retained the receptor specificity of RD114 and will therefore prove useful in receptor characterization.

Animals↗
PubMed1992

Cytokine modulation of cell growth and role in tumour therapy.

Cytokines are a group of secreted proteins which act as intracellular signals co-ordinating the growth and function of cells in the haematopoietic systems. Despite often overlapping functions they appear to have evolved separately but their receptors do share several features suggesting a common ancestor. Taking interleukin 2 (IL2) as an example we discuss the mechanisms involved with the regulation of IL2, the interleukin 2 receptor (IL2R) and their modes of action. Finally we discuss the various aspects of cytokines which allow their use as antitumour agents.

Adjuvants, Immunologic↗
PubMed1992

Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus.

Pseudotypes of gibbon ape leukemia virus/simian sarcoma-associated virus (GALV/SSAV) and feline leukemia virus subgroup B (FeLV-B) have been constructed by rescuing a Moloney murine leukemia virus vector genome with wild-type GALV/SSAV or FeLV-B. The resulting recombinant viruses utilized core and envelope proteins from the wild-type virus and conferred resistance to growth in L-histidinol upon infected cells by virtue of the HisD gene encoded by the vector genome. They displayed the host range specificity of the rescuing viruses and could be neutralized by virus-specific antisera. Receptor cross-interference was observed when the GALV/SSAV or FeLV-B pseudotypes were used to superinfect cells productively infected with either GALV/SSAV or FeLV-B. Although murine cells are resistant to FeLV-B infection, murine cells expressing the human gene for the GALV/SSAV receptor became susceptible to FeLV-B infection. Therefore GALV/SSAV and FeLV-B utilize the same cell surface receptor.

3T3 Cells↗
PubMed1992

Transformation-dependent expression of interleukin genes delivered by a recombinant parvovirus.

The prototype strain of minute virus of mice [MVM(p)] is an autonomous parvovirus with a tropism for cells expressing a neoplastically transformed phenotype. To generate gene transfer vectors for tumor-specific gene expression, human interleukin-2 (IL-2) and murine interleukin-4 (IL-4) genes were cloned under the control of the p38 late promoter of MVM(p). Upon transfection into permissive cells, the recombinant MVMIL2 or MVMIL4 DNA was excised, amplified, and, in the presence of a helper plasmid, packaged into recombinant viral particles. The recombinant viruses were able to transfer fully functional IL-2 and IL-4 genes to permissive target cells and retained the oncotropic host range properties of the parental virus. Following infection with MVMIL2, nontransformed fibroblasts of rodent (FR3T3) or human (MRC-5) origin produced minimal IL-2 compared with the high levels of IL-2 production observed in their transformed derivatives (FREJ4 and MRC-5V1).

Cell Line, Transformed↗
PubMed1992

The human immunodeficiency virus tat protein increases the transcription of human Alu repeated sequences by increasing the activity of the cellular transcription factor TFIIIC.

The HIV Tat protein is able to upregulate the transcription by RNA polymerase III of cotransfected or endogenous cellular Alu-repeated sequences in both HeLa and Jurkat T cells. This effect is mediated by an increase in the activity of transcription factor TFIIIC, which binds to the B box in the RNA polymerase III Alu promoter. This is the first example of an effect of the Tat protein on the transcription of a cellular gene or on the activity of a cellular transcription factor. The significance of this effect for the life cycle of HIV and its interaction with infected cells is discussed.

Actins↗
PubMed1991

Decreased tumorigenicity of a transplantable rat sarcoma following transfer and expression of an IL-2 cDNA.

The notion that tumour-cell-derived IL-2 might lead to paracrine stimulation of the host anti-tumour response was tested in a transplantable rat sarcoma model. Three HSNLV clones induced to secrete different amounts of human IL-2 following retroviral gene transfer showed reduced tumorigenicity and metastatic potential in athymic (nu/nu) and immunocompetent syngeneic rats which was directly related to the level of IL-2 secretion. In contrast, the tumorigenicity of HSNLV clones secreting a biologically inactive form of IL-2 (IL-2Lys20) was unaltered. T-lymphocyte-mediated rejection of ZipI, the highest IL-2 producer, was demonstrated histologically in hooded rats and infiltrating mononuclear cells were also observed in ZipI tumours growing in athymic rats. Tumours derived from IL-2-secreting HSNLV showed reduced or absent IL-2 secretion in immunocompetent rats, sometimes with associated loss of the IL-2 provirus, but continued to secrete IL-2 in nude rats. The host response to Moloney-helper-virus-infected HSNLV was also examined and the results represent a cautionary note to those undertaking experiments of a similar nature.

Animals↗
PubMed1991

A murine cell line producing HTLV-I pseudotype virions carrying a selectable marker gene.

A murine cell line, EH, expressing the gag and pol proteins of Moloney murine leukemia virus (Mo-MLV) as well as an Mo-MLV recombinant genome with a selectable marker (histidinol dehydrogenase), was transfected with a plasmid coding for the gene of the human T-cell leukemia virus type I (HTLV-I) envelope precursor (gp62), placed under the control of the human cytomegalovirus immediate early promoter. One clone, T. 14, was recovered, in which gp62 RNA and protein were detected. Supernatant from this clone transferred the HisD gene to a panel of cell lines which express receptors for HTLV-I, but was unable to pass the marker gene to cells which do not express receptors. The colony-forming units were sensitive to HTLV-I receptor interference and to specific neutralization by anti-HTLV-I serum. These data show that hybrid virions were produced in which the envelope proteins of HTLV-I had pseudotyped Mo-MLV capside particles containing a selectable recombinant viral genome.

Animals↗
PubMed1991

A recombinant HIV provirus is synergistically activated by the HIV Tat protein and the HSV IE1 protein but not by the HSV IE3 protein.

To study the effects of regulatory proteins encoded by herpes viruses on the HIV long terminal repeat (LTR) in the presence or absence of HIV-encoded regulatory products, we prepared a proviral construct containing 5' and 3' HIV LTR, but lacking the coding sequences of any HIV proteins. This construct allowed the effects of herpesvirus regulatory proteins on the HIV LTR to be assessed in a construct similar to the HIV provirus whilst also allowing their interactions with HIV-encoded regulatory proteins to be studied. In this system, the herpes simplex virus (HSV) protein IE1 (ICPO) but not the IE3 (ICP4) protein can activate the HIV LTR, whereas both proteins are active on a single plasmid-borne HIV LTR. Although the activation of the LTR by IE1 is strongly stimulated by the HIV Tat protein, it can also be observed in the absence of Tat, indicating that HSV infection via IE1 has the potential to activate an entirely silent, latent HIV provirus.

Animals↗
PubMed1991

A randomized comparison of umbilical vein and intravenous oxytocin during the puerperium.

Oxytocin was administered in a randomized fashion via either the umbilical or maternal intravenous route. Women who received intraumbilical oxytocin had significantly greater calculated blood loss compared with those who received peripheral administration (P = .01). This greater blood loss was confirmed by a decrease in hematocrit and hemoglobin concentrations after delivery. There was no difference between the groups in the length of the third stage of labor. The incidence of fetomaternal transfusion was higher in the intraumbilical group (P = .07). We conclude that intraumbilical oxytocin is no more beneficial than peripheral administration.

Adult↗
PubMed1990

Evidence that the level of the p55 component of the interleukin (IL) 2 receptor can control IL 2 responsiveness in a murine IL 3-dependent cell.

Although the role of interleukin 2 (IL 2) in mature T lymphocyte function is well documented, its effect on hematopoietic progenitor cells is less well characterized. Here we have used recombinant retroviruses to transduce and express a cDNA clone encoding the p55 component of the human IL 2 receptor (h-p55), in a murine IL 3-dependent cell line, BAF3. While the parental cells do not respond to IL 2, the h-p55-expressing cells proliferate upon treatment with recombinant IL 2 after an initial lag period. The responsiveness of individual cell clones is correlated with their level of h-p55 expression, and can be inhibited by Tac monoclonal antibody. Furthermore, growth at limiting IL 2 concentrations selects a subset of cells expressing higher h-p55 levels from a bulk population. Detailed 125I-labeled IL 2 binding analysis on the highest h-p55-expressing clone detects the presence of 200 high-affinity (KD = 25 pM) IL 2 receptors. We therefore propose that the level of h-p55 expression governs the formation of high-affinity receptors, and hence IL 2 responsiveness in BAF3 cells.

Animals↗