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Biomedical subjects

J H Phillips

Publications and source records attributed to J H Phillips.

At least 19 recordsLinked to original sources
PubMed1992

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.

Adrenal Medulla↗
PubMed1992

Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes.

NK cells have been defined as CD3-, CD16+, and/or CD56+ lymphocytes that mediate MHC-unrestricted cytotoxicity against certain tumors and virus-infected cells. Although CD3 epsilon transcripts have been detected in some NK clones, it has generally been thought that NK cells do not express CD3 proteins other than zeta which is associated with CD16 (Fc gamma RIII). We demonstrate that adult peripheral blood NK cell lines and clones express cytoplasmic CD3 epsilon proteins, but not CD3 delta or gamma. CD3 epsilon proteins were detected by immunoprecipitation, Western blot analysis, and immunofluorescence using antiserum directed against the cytoplasmic domain of CD3 epsilon. Although resting, adult peripheral blood NK cells have essentially undetectable levels of CD3 epsilon protein, expression was increased substantially after activation. In contrast to adult NK cells, NK cell clones established from human fetal liver express CD3 gamma, delta, and epsilon protein subunits that associate and form CD3 epsilon, gamma and CD3 epsilon, delta complexes in the cytoplasm, but are apparently unable to be transported to the cell surface. These results indicate that expression of CD3 gamma delta epsilon subunits is not restricted to T lymphocytes and supports the possibility that NK and T cells may be derived from common origins.

Age Factors↗
PubMed1992

Human fetal liver-derived CD7+CD2lowCD3-CD56- clones that express CD3 gamma, delta, and epsilon and proliferate in response to interleukin-2 (IL-2), IL-3, IL-4, or IL-7: implications for the relationship between T and natural killer cells.

Cells from fetal liver or fetal and adult bone marrow that are membrane (m)CD3 negative and have not rearranged TCR genes but express CD3 proteins in their cytoplasm are considered to be committed T-cell progenitors. Recent findings question whether CD3 is T-cell specific because fetal natural killer (NK) cells have been shown to express cCD3 delta and epsilon proteins. To further examine the relationship between T and NK cells, we generated mCD3-cCD3+ clones from fetal liver. Two stable clones, FL412 and FL508, isolated from different donors, were selected on the basis of absence of the NK cell marker CD56. These clones shared a common phenotype of CD7+CD2lowCD3-CD4-CD8-CD5-CD6-CD11b+CD16-CD56 -. Like fetal NK clones, these clones expressed cytoplasmic CD3 delta and epsilon transcripts and proteins. However, the clones exhibited no or very low levels of cytotoxic activity against K562, JY, or Daudi, which were lysed efficiently by fetal NK clones. TCR beta, gamma, and delta genes in these clones were in germline configuration. Furthermore, both FL412 and FL508 responded not only to interleukin-2 (IL-2), IL-4, or IL-7, but also to IL-3 with proliferation. These results suggest that FL412 and FL508 retained some characteristics of a putative T or NK precursor in the fetal liver and may be useful for analyses of this poorly defined cell type.

Antigens, CD↗
PubMed1992

Involvement of CD28 in MHC-unrestricted cytotoxicity mediated by a human natural killer leukemia cell line.

NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.

Antigens, CD↗
PubMed1992

Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins.

Natural killer (NK) cells have been defined as CD3 epsilon-, CD16+ and/or CD56+ lymphocytes that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against certain tumors and virus-infected cells. Unlike T lymphocytes, NK cells do not rearrange or productively express T cell antigen receptor genes. Moreover, NK cells from adults have been reported to not express CD3 gamma, delta, or epsilon proteins on the cell surface or in the cytoplasm. Nonetheless, NK cells have been shown to share a number of antigenic and functional similarities to T cells that suggest the possibility of common origins. In this report, we demonstrate that functional NK cells exist in liver at early stages of human embryonic development. Freshly isolated fetal NK cells mediated MHC-unrestricted cytotoxicity against NK-sensitive targets and acquired the ability to lyse NK-resistant tumors after overnight culture in interleukin 2. Unlike adult NK cells, freshly isolated fetal liver NK cells and clones derived from these cells, as well as a subset of cord blood NK cells, express substantial levels of CD3 delta and CD3 epsilon proteins in the cytoplasm. Expression of CD3 epsilon and CD3 delta transcripts and cytoplasmic proteins in fetal NK clones was confirmed by polymerase chain reaction and Western blot analysis. These findings support the concept that NK and T cells may arise from a common progenitor that expresses components of the CD3 complex. Alternatively, it is possible that the cytoplasmic CD3 delta, epsilon+ fetal NK cells represent a distinct subpopulation of NK cells that is predominant in the fetus, but replaced by the cytoplasmic CD3 delta,epsilon- adult NK cell population after embryogenesis.

Antigens, CD↗
PubMed1992

CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes.

Engagement of the CD3/T cell antigen receptor complex on small, resting T cells is insufficient to trigger cell-mediated cytotoxicity or to induce a proliferative response. In the present study, we have used genetic transfection to demonstrate that interaction of the B7-BB1 B cell activation antigen with the CD28 T cell differentiation antigen costimulates cell-mediated cytotoxicity and proliferation initiated by either anti-CD2 or anti-CD3 monoclonal antibody (mAb). Moreover, a B7-negative Burkitt's lymphoma cell line that fails to stimulate an allogeneic mixed lymphocyte response is rendered a potent stimulator after transfection with B7. The mixed leukocyte reaction proliferative response against the B7 transfectant is inhibited by either anti-CD28 or B7 mAb. We also demonstrate that freshly isolated small, resting human T cells can mediate anti-CD3 or anti-CD2 mAb-redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma transfected with human B7. These preexisting cytotoxic T lymphocytes in peripheral blood are present in both the CD4 and CD8 subsets, but are preferentially within the CD45RO+ "memory" population. While small, resting T cells apparently require costimulation by CD28/B7 interactions, this requirement is lost after T cell activation. Anti-CD3 initiates a cytotoxic response mediated by in vitro cultured T cell clones in the absence of B7 ligand. The existence of functional cytolytic T cells in the small, resting T cell population may be advantageous in facilitating rapid responses to immune challenge.

Animals↗
PubMed1992

The developmental relationship between NK cells and T cells.

NK cells and T cells share many features, suggesting that they have a common origin. Here, Lewis Lanier and colleagues describe recent studies of fetal ontogeny and thymic development that provide support for this hypothesis, and propose an integrated scheme for NK-cell and T-cell development.

Animals↗
PubMed1992

Natural killer cells.

Although the antigen-specific receptors of T and B lymphocytes have been characterized, the receptors used by natural killer cells to recognize normal cells, tumors, and virus-infected cells have remained elusive. Recently, experimental systems have been developed to identify these structures, and candidate signal-transducing molecules have been proposed.

Animals↗
PubMed1992

CD28 co-stimulation of T-cell-mediated cytotoxicity.

Co-stimulation via the CD28 pathway permits small, resting human peripheral-blood T lymphocytes to mediate anti-CD3 monoclonal antibody (MAb) "re-directed" cytotoxicity. The effector cells are contained with the "memory" population of T lymphocytes, identified by expression of the CD45RO differentiation antigen. In this article, we review the requirements for initiating a cytolytic response and speculate on the physiological consequences of this process.

Animals↗
PubMed1992

Current concepts in the use of bone grafts in facial fractures. Basic science considerations.

In choosing the ideal donor site for bone replacement, attention must be given to the recipient site's revascularization potential and mechanical needs. Based on histologic differences in architecture as related to these two factors, the most appropriate graft type may be harvested. Future potential exists for bone replacement by distraction osteogenesis without the need for remote site harvesting. This may provide for the most identical bone replacement.

Biomechanical Phenomena↗
PubMed1992

A stereotactic system for guiding complex craniofacial reconstruction.

A stereotactic system has been designed to address the problem of achieving symmetry in complex and extensive craniofacial defects. Preliminary testing suggests that such a system, which allows for the intraoperative application of preoperative CT planning, will be useful in guiding the reconstruction of congenital or acquired bony time, is being used to investigate the correlation of intraoperative globe position following enophthalmos correction with long-term outcome, particularly as it relates to the size and location of the orbital defect, and the timing of the procedure.

Adult↗
PubMed1991

A novel beta 4, alpha 6 integrin-associated epithelial cell antigen involved in natural killer cell and antigen-specific cytotoxic T lymphocyte cytotoxicity.

Efficient immune responses require interactions between cell adhesion molecules on lymphocytes and counter-receptors on antigen presenting cells or target cells. While target-specific receptors or ligands have not been identified for natural killer (NK) cells, cell adhesion molecules have been implicated in the interaction between NK cell effectors and tumor cell targets. Herein, we describe monoclonal antibodies (mAbs) against a carcinoma cell line that efficiently block the cytolytic activity of interleukin 2-activated NK cell lines and clones. L280 mAb reacts with secretory epithelial cells in normal human tissues, but does not react with hematopoietic cells or other tissue types. Biochemical analysis revealed that L280 mAb immunoprecipitates the beta 4, alpha 6 integrin, as well as a novel 98-kD glycoprotein, and probably reacts with a carbohydrate epitope on these molecules. Involvement of the L280 antigen in cellular immunity is not restricted to NK cell-mediated cytotoxicity. L280 mAb also efficiently inhibits alloantigen-specific cytotoxicity against Colo-205 cells mediated by human histocompatibility leukocyte antigen (HLA)-A2 alloantigen specific alpha beta-TCR+ and gamma delta-TCR+ cytotoxic T lymphocyte (CTL) clones. Additionally, we demonstrate that L280 mAb blocks cytotoxicity mediated by influenza peptide-specific HLA-restricted CTL clones. These data indicate that the antigen recognized by L280 mAb is important in both NK and CTL function, and that an as yet unidentified receptor for this epithelial antigen is present on both NK and T lymphocytes. The restricted expression of L280 antigen indicates that this molecule may be important in immune reactions in epithelial tissues.

Adenocarcinoma↗
PubMed1991

Involvement of a metalloprotease in spontaneous and phorbol ester-induced release of natural killer cell-associated Fc gamma RIII (CD16-II).

Two genes encode the CD16 low affinity IgG FcR. CD16-I (Fc gamma RIII-1) is expressed on PMN as a phosphatidylinositol-glycan anchored glycoprotein. CD16-II (Fc gamma RIII-2) is expressed on NK cells and macrophages as a transmembrane glycoprotein associated with CD3 zeta or Fc epsilon RI-gamma. NK cells spontaneously release soluble CD16-II from the cell surface and this is enhanced by activation with phorbol ester. In this study, we demonstrate that a metalloprotease is involved in the spontaneous and PMA-induced release of CD16-II from NK cells. 1,10-phenanthroline, an inhibitor of Zn(2+)-dependent metalloproteases, efficiently inhibits CD16-II release. 1,7-phenanthroline, an inactive analogue that doesn't chelate Zn2+ or other divalent metal cations, and inhibitors of serine proteases do not affect spontaneous or PMA-induced release of CD16-II. Murine P815 mastocytoma cells transfected with human CD16-II cDNA shed membrane CD16, and 1,10-phenanthroline inhibits this process. P815 transfectants expressing CD16-II molecules with truncated cytoplasmic domains also release soluble receptors, indicating that the cytoplasmic segment of CD16-II is not required for interaction with the protease or the cytoskeleton. By contrast, 1,10-phenanthroline does not inhibit PMA-induced release of CD16-I glycoprotein from PMN, indicating a different mechanism of release for this phosphatidylinositol-glycan anchored molecule. Prior studies have demonstrated that NK cells are activated via the inositol phosphate pathway after engagement of CD16-II by immune complexes or Ig-coated tumor cell targets. A membrane metalloprotease with substrate specificity for CD16-II that is activated by PKC stimulation may provide a mechanism for releasing the immune complex or target from the effector cells and halting signal transduction.

Alkaloids↗
PubMed1991

Glycosylation and transmembrane topography of bovine chromaffin granule p65.

The bovine homologue of p65, a calmodulin-binding protein located in the membranes of synaptic vesicles and endocrine secretory granules, has been studied by the use of monoclonal antibodies directed against this antigen and against dopamine beta-mono-oxygenase. The protein (apparent molecular mass 67 kDa; pI = 5.5-6.2) is partially degraded by treatment with neuraminidase or endoglycosidase F. Trypsin treatment of intact adrenal chromaffin granules or of granule membranes releases a soluble 39 kDa fragment of p65 which corresponds to the whole of its cytoplasmic domain. This domain contains both the epitope for the monoclonal antibody cgm67 and the calmodulin-binding site. The 20 amino acids at the N-terminus of this fragment are identical to part of the rat p65 sequence.

Amino Acid Sequence↗
PubMed1991

Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.

Antigens, CD↗
PubMed1991

Analysis of Fc gamma RIII (CD16) membrane expression and association with CD3 zeta and Fc epsilon RI-gamma by site-directed mutation.

Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.

Antigens, CD↗
PubMed1991

Platelet-induced expression of Fc gamma RIII (CD16) on human monocytes.

Monocytes constitutively express Fc receptor (FcR) for IgG type I (CD64) and type II (CD32), but not type III (CD16). Prior studies have indicated that in vitro culture of monocytes results in spontaneous induction of CD16, but this phenomenon has been variable and the mechanism unexplained. Here, we demonstrate that activated platelets are responsible for induction of CD16 on monocytes, as a consequence of TGF-beta release. Local release of TGF-beta by activated platelets at sites of tissue damage may induce CD16 on infiltrating or resident monocytes that in turn facilitate the function of these effector cells. The FcR on these CD16+ monocytes is functionally competent, and enables the monocytes to kill murine anti-CD16 hybridoma cells. CD16 expressed on platelet-activated monocytes is structurally similar to the transmembrane-anchored CD16-II polypeptide expressed on natural killer (NK) cells. However, whereas CD16-II on NK cells is co-associated with CD3 zeta, we were unable to detect CD3 zeta transcript or protein in monocytes. Transcripts for the gamma subunit of the high-affinity IgE FcR (Fc epsilon RI-gamma) were detected in monocytes, and presumably gamma proteins are co-associated with CD16-II in these cells. Nucleotide sequence analysis of Fc epsilon RI-gamma cDNA derived from both NK cells and cultured monocytes indicated identity with the structure previously cloned from basophils. Since CD16+ monocytes can kill anti-CD16 hybridoma cell targets in the absence of CD3 zeta, these results indicate that CD3 zeta is not essential for signal transduction in CD16-II-mediated cytotoxicity.

Antibody-Dependent Cell Cytotoxicity↗