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Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 109 records · Page 6Linked to original sources
PubMed1993

Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells.

Cassette and deletion mutagenesis were used to analyze the function of the amphipathic alpha-helices in the transmembrane domain of DAB389-interleukin-2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor. We demonstrate that the in-frame deletion of 60 amino acids, from Asn204 to Glu263 in DAB389-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp208 to Ser220 and Ala244 to His258 are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity. DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin. Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB389-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess "wild type" catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB389-IL-2 binds to the IL-2 receptor. It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP-ribosyltransferase from DAB389-IL-2 to the cytosol of target cells.

Amino Acid Sequence↗
PubMed1993

Activation and enhanced contact of human T-lymphocytes with autologous red blood cells are required for their stable adherence at 37 degrees.

The adherence of human red blood cells (RBC) to autologous T-cells does not occur in the body, and in vitro is elicited at 4 degrees. Autologous E-rosetting at 37 degrees has not previously been described. In this work, lymphocyte-RBC adherence has been studied in mixed leukocyte-RBC cultures and in whole blood from healthy donors. Vital, cytochemical and electron microscopic studies have shown that T-cells may form stable E-rosettes with autologous RBC at 37 degrees. As in the previously reported cold-dependent reversible rosetting, stable rosetting is mediated by the erythrocyte LFA3 and lymphocyte CD2 molecules. Uniquely, this phenomenon requires both T-cell activation and an enhanced contact between the T-cell and RBC membranes. These requirements were met by exposure of cell cultures to: (1) PHAE, the erythroagglutinating component of PHAP, or (2) to either non-erythroagglutinating mitogens, PHAL, Con A, OKT3 or SEA, or to antigens of typhus group rickettsiae or salmonellae, provided that the RBC membrane was desialyted. Cultures derived from individuals seropositive to rickettsiae or vaccinated with salmonellae demonstrated the adherence phenomenon after antigen exposure when neuraminidase was present in the culture medium. The system 2 described here can be used as a diagnostic tool for defining activated T-cells and T-cell clones with the memory to antigens capable of inducing cell-mediated immunity.

Cell Adhesion↗
PubMed1993

Diarrhoeal disease: current concepts and future challenges. Rapid detection of enteric infections by measurement of enteric pathogen-specific antibody secreting cells in peripheral blood.

Enteric infections and diarrhoeal disease are important causes of morbidity and mortality. Infections are often difficult to diagnose, especially when access to sophisticated laboratory facilities is limited. In an attempt to develop a rapid method of diagnosis, which might not require an advanced laboratory, advantage was taken of the observation that precursors of lymphocytes secreting immunoglobulin A (IgA), generated in response to mucosal stimulation with an antigen, are transiently present in peripheral blood. Detection of cells in peripheral blood bearing IgA specific for a pathogenic microbe should indicate current infection. This hypothesis was tested using peripheral blood mononuclear cells collected from volunteers who were clinically 'normal', had experimentally induced shigellosis, had naturally acquired shigellosis, or had naturally acquired typhoid fever. The method was sensitive for detection of Salmonella typhi infection and less sensitive for detection of Shigella infection. The antibody secreting cell procedure has good potential as a rapid, simple diagnostic procedure if applied during the acute phase of infection.

Adult↗
PubMed1993

Acute lower gastrointestinal bleeding.

Lower gastrointestinal bleeding ranges from occult blood loss to massive hemorrhage and shock. There are many causes but diverticulitis and angiodysplasia remain the most common sources of major hemorrhage. This article emphasizes the cause and evaluation of moderate to severe acute lower gastrointestinal bleeding.

Acute Disease↗
PubMed1993

Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6.

We have used site-directed and in-frame deletion mutational analysis in order to explore the structural features of the IL-6 portion of the diphtheria toxin-related interleukin-6 (IL-6) fusion toxin DAB389-IL-6 that are essential for receptor-binding and subsequent inhibition of protein synthesis in target cells. Deletion of the first 14 amino acids of the IL-6 component of the fusion toxin did not alter either receptor binding affinity or cytotoxic potency. In contrast, both receptor binding and cytotoxic activity were abolished when the C-terminal 30 amino acids of the fusion toxin were deleted. In addition, we explored the relative role of the disulfide bridges within the IL-6 portion of DAB389-IL-6 in the stabilization of structure required for receptor-binding. The analysis of mutants in which the substitution of either Cys440, Cys446, Cys469 or Cys479 to Ser respectively, demonstrates that only the disulfide bridge between Cys469 and Cys479 is required to maintain a functional receptor binding domain. In addition, the internal in-frame deletion of residues 435-451, which includes Cys440 and Cys446, was found to reduce, but not abolish receptor binding affinity. These results further demonstrate that the disulfide bridge between Cys440 and Cys446 is not essential for receptor-binding. However, the reduced cytotoxic potency of DAB389-IL6(delta 435-451) suggests that the conformation and/or receptor binding sites associated with this region of the fusion toxin is/are important for maintaining the wild type receptor binding affinity and cytotoxic potency.

Base Sequence↗
PubMed1993

Differential sensitivity of human myeloma cell lines and normal bone marrow colony forming cells to a recombinant diphtheria toxin-interleukin 6 fusion protein.

The cytotoxicity of a recombinant interleukin 6 (IL-6)-diphtheria toxin (DT) fusion protein towards human myeloma cell lines was investigated. DAB389-IL-6 inhibited protein synthesis and methylcellulose colony formation by U266 myeloma cells. In the clonogenic assay, the fusion protein approached the level of cytotoxicity achieved by native DT. The specificity of killing by DAB389-IL-6 was demonstrated by inhibition of cytotoxicity by a molar excess of free rhIL-6. The effect of DAB389-IL-6 on colony formation by six OCI-My cell lines was assessed. Similar to U266 cells, colony growth by the OCI-My 5 and -My 2 cell lines was inhibited in a simple dose dependent manner. However, a biphasic effect was observed for the IL-6 dependent OCI-My 4 cells; DAB389-IL-6 stimulated colony formation at low (< or = 10(-11) M) concentrations, yet was inhibitory at higher doses. Three other cell lines whose growth was not altered by IL-6 were relatively unaffected by DAB389-IL-6, despite their sensitivity to native DT. Flow cytometric analysis for IL-6 receptor expression using phycoerythrin-conjugated IL-6 demonstrated specific binding sites on both DAB389-IL-6 sensitive and certain insensitive cell lines, suggesting that other factors in addition to the expression of IL-6 receptors are involved in killing by the fusion toxin. Despite evidence for a role of IL-6 in myeloid cell development, normal bone marrow was insensitive to the IL-6 fusion toxin. In cultures containing both normal bone marrow and U266 cells DAB389-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth. From these experiments we conclude that DAB389-IL-6 is specifically cytotoxic towards a subset of IL-6-responsive human myeloma cell lines and may be useful, in some cases, in the selective elimination of tumour cells from mixed populations of normal and malignant cells.

Cells, Cultured↗
PubMed1993

Interleukin-2 receptor-directed therapies: antibody-or cytokine-based targeting molecules.

With the exception of certain hematologic malignancies, the high affinity interleukin-2 (IL-2) receptor is only transiently expressed during the brief antigen-triggered proliferative burst of lymphocytes. Hence, we wondered whether administration of anti-IL-2 receptor (IL-2R) monoclonal antibody (mAb) or chimeric IL-2 toxins would provide a utilitarian way to achieve immunosuppression aimed directly at activated lymphocytes, or whether this approach could be used to treat IL-2R+ leukemia/lymphoma. Studies in preclinical autoimmune and transplant models indicate that this approach can be effective. The results of open, uncontrolled studies provide preliminary evidence that a chimeric IL-2 toxin is well tolerated at doses that may induce improvement in patients with IL-2R+ leukemia/lymphoma, as well as in patients with refractory rheumatoid arthritis or new-onset diabetes mellitus.

Animals↗
PubMed1993

The natural history of amyotrophic lateral sclerosis.

Using 42 strength and functional assessments recorded monthly, the natural history of amyotrophic lateral sclerosis (ALS) is described in 167 patients (98 men, 67 women) followed in five medical centers in the western United States. The mean age at onset was 57.4 years, and symptoms were present for 2.64 years before study entry. Although there was a highly variable rate of decline within the group of patients, there were no differences in rate of decline by age or gender. Older patients and women were weaker on entry. Forty-eight patients died during the study. The median survival was 4.0 years for the study cohort but 2.1 years for newly diagnosed cases. Decline in pulmonary function most closely correlated with death. Our results emphasize the importance of considering clinical variability in planning clinical trials. One possible strategy is to identify and stratify patients by rate of decline in pulmonary function since prospectively identifying homogeneous subgroups allows investigators to substantially reduce sample size in therapeutic trials.

Adult↗
PubMed1993

Cytotoxicity of a recombinant diphtheria toxin-granulocyte colony-stimulating factor fusion protein on human leukemic blast cells.

Granulocyte colony-stimulating factor (G-CSF) is a potent stimulator of the growth of normal and malignant hematopoietic cells and synergizes with other factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The action of G-CSF is mediated through a specific membrane receptor, however it is not clear if all of the effects of G-CSF are direct or indirect. As a step towards addressing this problem, a recombinant diphtheria toxin (DT)-related human G-CSF fusion protein has been constructed and purified from E. coli. The 70,000 dalton chimeric protein has immunologic determinants characteristic of both DT and G-CSF. At high concentrations, DAB486-G-CSF is cytotoxic towards G-CSF-dependent OCI/AML1 cells, but not factor independent OCI/AML3 cells; colony formation by G-CSF-responsive leukemic blasts from a patient with acute myeloblastic leukemia (AML) was also inhibited. The G-CSF fusion toxin displayed ADP-ribosyltransferase activity in a cell-free system. Genetic conjugation of G-CSF to an enzymatically inactive DT mutant, CRM197, resulted in a 200-fold reduction in the ability of G-CSF to stimulate normal bone marrow colony formation. These results suggest that fusion of G-CSF to DT sequences interferes with some of the activity but not the specificity of the ligand binding domain of the molecule. Nevertheless, DAB486-G-CSF may be included with the increasing number of other toxin-hormone fusion proteins whose toxicity is directed towards specific receptor-bearing cells, and may represent a novel approach towards the study and treatment of leukemia.

ADP Ribose Transferases↗
PubMed1993

Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes.

Experiments were conducted to determine whether West Nile (WN) virus was transmitted vertically by colonized strains of Aedes albopictus, Ae. aegypti, and Culex tritaeniorhynchus. Female mosquitoes were infected by intrathoracic inoculation with WN virus, and the F1 progeny were tested for virus by the fluorescence antibody technique and the newborn mouse assay. Each of the three mosquito species transmitted WN virus to F1 adults derived from immature forms reared at 26 degrees C. The minimal filial infection rate (MFIR) ranged from 1:124 to 1:138 for Ae. albopictus, from 1:62 to 1:172 for Ae. aegypti, and from 1:325 to 1:859 for Cx. tritaeniorhynchus. The MFIR for Cx. tritaeniorhynchus reared at 20 degrees C was 1:213 for larvae and 1:390 for pupae, and 1:208 for larvae and 1:554 for pupae reared at 26 degrees C. These data are the first reported evidence of vertical transmission of WN virus by mosquitoes, and therefore warrant further studies to determine whether vertical transmission occurs among WN viral-infected mosquitoes in nature.

Aedes↗
PubMed1992

Binding of the metalloregulatory protein DtxR to the diphtheria tox operator requires a divalent heavy metal ion and protects the palindromic sequence from DNase I digestion.

Transcription of the corynebacteriophage diphtheria tox operon has been shown to be regulated through a corynebacterial determined factor DtxR. The dtxR gene has been recently cloned and expressed in Escherichia coli, and shown to regulate the expression of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion. Tao et al. (Tao, X., Boyd, J., and Murphy, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5897-5901) have recently shown by gel mobility shift assay that the binding of DtxR to the tox operator requires a divalent heavy metal ion, as well as the 27-base pair interrupted palindromic sequence. We now show the activation of DtxR in the presence of Co2+, Fe2+, or Mn2+ results in the protection of a 33- and 27-nucleotide region on the coding and non-coding strand from DNase I digestion, respectively. DtxR is also activated in the presence of Ni2+; however, this metalloregulatory factor is only weakly activated by Zn2+. The diphtheria tox regulatory region protected from DNase I digestion in the presence of activated DtxR encompasses the 27-base pair interrupted palindromic sequence.

Bacterial Proteins↗
PubMed1992

Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.

Binding, Competitive↗
PubMed1992

Specific binding of the diphtheria tox regulatory element DtxR to the tox operator requires divalent heavy metal ions and a 9-base-pair interrupted palindromic sequence.

The structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages; however, the regulation of tox expression is controlled by a Corynebacterium diphtheriae-encoded regulatory element, dtxR. The molecular cloning and sequence analysis of dtxR was recently described. Previous studies have suggested that DtxR-mediated regulation of the diphtheria tox operator involves the formation of an iron-repressor complex, which specifically binds to the tox operator. We have expressed and purified DtxR from recombinant Escherichia coli. Immunoblot analysis shows DtxR to be a single M(r) 28,000 protein band in both recombinant E. coli and the C7(-) and C7hm723(-) strains of C. diphtheriae. In addition, we demonstrate that the binding of DtxR to a diphtheria tox promoter/operator probe requires the addition of Mn2+ to the reaction mixture; however, binding may be blocked by addition of the chelator 2,2'-dipyridyl, anti-DtxR antiserum, and excess unlabeled probe to the reaction mixture. Deletion of one of the 9-base-pair inverted repeat sequences from the tox operator results in a loss of DtxR binding. The results presented here demonstrate that regulation of diphtheria toxin expression by DtxR requires direct interaction between this regulatory factor and the tox operator in the presence of a divalent heavy metal ion.

Bacterial Proteins↗
PubMed1992

Interaction of an alpha-melanocyte-stimulating hormone-diphtheria toxin fusion protein with melanotropin receptors in human melanoma metastases.

A hybrid toxin targeted to melanotropin receptors and selectively cytotoxic to melanoma cell lines in vitro has recently been developed. The toxin, a recombinant fusion protein (designated DAB389-MSH), contains the peptide sequences of alpha-melanocyte-stimulating hormone (alpha-MSH) and the catalytic (cytotoxic; Fragment A) and lipophilic (part of Fragment B) domains of diphtheria toxin. In the present study, binding of DAB389-MSH to melanotropin receptors in biopsy specimens of human and mouse melanoma metastases was assessed by measuring its ability to inhibit binding of a radiolabeled, superpotent analogue of alpha-MSH (125I-[Nle4,D-Phe7]-alpha-MSH; 125I-NDP-MSH) and comparing its potency in this system with those of the established ligands NDP-MSH and alpha-MSH. Radioligand binding to tissue sections in vitro was localized and quantified by autoradiography and image analysis. DAB389-MSH inhibited binding of 125I-NDP-MSH to experimental murine B16-F1C23 melanoma metastasis tissue and to melanoma metastases of three patients. In both mouse and human melanoma tissues, concentration-response relationships for DAB389-MSH-mediated inhibition of 125I-NDP-MSH binding were parallel, and its maximal effects were comparable in magnitude, to those of NDP-MSH and alpha-MSH. Half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 0.63 nM; alpha-MSH, 3.14 nM; and DAB389-MSH, 10.1 nM. In human melanoma tissues, the respective half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 1.80 nM; alpha-MSH, 2.43 nM; and DAB389-MSH, 11.9 nM. Taken together, these results suggest that NDP-MSH, alpha-MSH, and DAB389-MSH bind to a common melanotropin receptor in human metastatic melanoma cells. Since previous work has shown that melanotropin receptors are detectable in melanoma metastases of about 80% of human patients, malignant melanoma cells of many patients may be susceptible to killing by the melanotropin receptor-targeted cytotoxin DAB389-MSH.

Animals↗
PubMed1992

Interleukin 2 receptor targeted fusion toxin (DAB486-IL-2) treatment blocks diabetogenic autoimmunity in non-obese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) is strikingly similar in the non-obese diabetic (NOD) mouse and humans. In IDDM, the systematic autoimmune destruction of insulin-producing beta cells within the pancreas is dependent on autoreactive T cells. This autoimmune process can be accelerated by transferring spleen cells from diabetic donors into irradiated syngeneic NOD mice. In a previous study we established that interleukin 2 receptor (IL 2R)-bearing cells propagated from pre-diabetic NOD mice promote IDDM. Therefore, we reasoned that specific elimination of IL 2R+ T cells should abort the diabetogenic process. T cell expressing IL 2R can be selectively destroyed with a diphtheria toxin-related IL 2 fusion protein (DAB486-IL-2). We set DAB486-IL-2 the challenging task of preventing fulminant IDDM accelerated by the adoptive transfer of diabetic spleen cells. Eight weeks after the adoptive transfer only 10% and 20% of NOD mice treated with 10 and 5 micrograms/day of DAB486-IL-2, respectively, became diabetic while 100% control mice (vehicle buffer) became diabetic within 5 weeks. A dose of 1 microgram/day of DAB486-IL-2 had no protective effect. Although the protection conferred by DAB486-IL-2 is not permanent, it is maintained for at least 4 weeks following cessation of treatment. Furthermore, even though these NOD mice do eventually become diabetic, the tempo of expression and severity of diabetes, as assessed by the level of hyperglycemia, is dramatically reduced. Although histologic examination of pancreas revealed minimal degree of mononuclear infiltrate within the islets in both groups, the vehicle control mice had fewer islets per section indicating many islets had already been destroyed. In addition, spleen cells from diabetic NOD mice which were pre-treated with DAB486-IL-2 (10 micrograms/day) for 1 week lost their ability to transfer disease. Taken together, these studies strongly support the concept that IL 2R-bearing T cells are essential for the induction of IDDM and suggest that DAB486-IL-2 would be a promising therapeutic approach in the treatment of human IDDM.

Animals↗
PubMed1992

A recombinant diphtheria toxin related human CD4 fusion protein specifically kills HIV infected cells which express gp120 but selects fusion toxin resistant cells which carry HIV.

A chimeric toxin made by a genetic fusion between the DNA encoding the 389 N-terminal amino acids of diphtheria toxin and that coding for the V1 and V2 domains of human CD4 (amino acids 1-178) was produced, purified and examined for ADP ribosylation activity, gp120 binding and effects on acutely and chronically HIV infected cells. The fusion toxin DAB389CD4 possesses enzymatic activity and binds to gp120. DAB389CD4 was found to kill CEM and U937 cells infected by HIV selectively and efficiently in a dose dependent manner, however, fusion toxin treatment did not eliminate the virus from acutely infected cell cultures. In addition, treatment of chronically infected cells with DAB389CD4 rapidly led to the appearance of HIV infected cells which were resistant to the chimeric toxin. The experimental results reported here suggest that the potential use of gp120 targeted cytotoxic agents for the treatment of HIV infection should be viewed with caution.

Adenosine Diphosphate Ribose↗
PubMed1992

Prolonged clearance is the primary abnormal reflux parameter in patients with progressive systemic sclerosis and esophagitis.

The purpose of this study is to determine if frequent reflux events from an incompetent LES or poor clearance from decreased peristalsis is the predominant abnormality in PSS patients with severe reflux esophagitis. Seven patients with both classic manometric findings of PSS and endoscopic findings of esophageal ulcerations and/or Barrett's esophagus were compared to nine patients with similar endoscopic findings but with no evidence of a connective tissue disorder. All patients underwent simultaneous intraesophageal pH monitoring and scintigraphy for a total of 40 min after a radiolabeled meal. Four of the PSS patients and all the non-PSS patients had simultaneous manometry. We found that PSS patients had significantly fewer reflux events (P less than 0.01), but the reflux events had significantly longer duration (P less than 0.01) compared to patients with similar severity of esophagitis and no connective tissue disease. We conclude that decreased smooth muscle peristalsis appears to be the primary contributor to acid exposure and esophageal injury in PSS.

Esophagitis, Peptic↗