PubMed Health⌕ Search

Biomedical subjects

K J Micklem

Publications and source records attributed to K J Micklem.

At least 19 recordsLinked to original sources
PubMed1991

HML-1 antigen on mucosa-associated T cells, activated cells, and hairy leukemic cells is a new integrin containing the beta 7 subunit.

The monoclonal antibodies HML-1, B-ly7 and Ber-ACT8 recognize intramucosal gut T lymphocytes, activated cells, and hairy cell leukemia. The antigen on hairy cells consists of three glycoproteins (160 kappa D, 130 kappa D and 105 kappa D unreduced; 145 kappa D and 120 kappa D reduced). These peptides have biochemical features reminiscent of integrins but we have shown by immunoprecipitation that they are not known integrin subunits. We have used a newly produced antibody (BP6) to purify this molecule and shown by N-terminal sequence analysis that the smallest subunit is the product of integrin beta 7 cDNA. This molecule is thus a new member of the integrin family of leucocyte adhesion proteins. Immunoprecipitation experiments indicate that the two larger subunits are recognized by HML-1, B-ly7 and Ber-ACT8.

Amino Acid Sequence↗
PubMed1990

Different isoforms of human FcRII distinguished by CDw32 antibodies.

The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.

Amino Acid Sequence↗
PubMed1989

KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections.

A new monoclonal antibody, KP1, raised against a lysosomal fraction of human lung macrophages, recognises a fixation-resistant epitope in a wide variety of tissue macrophages (such as Kupffer cells germinal centre, splenic, and lamina propria macrophages), and in granulocyte precursors. Its broad reactivity with cells of the mononuclear phagocytic lineage was established by testing on routinely processed samples of normal and reactive lymphoid tissues. Interdigitating reticulum cells were unstained or showed limited cytoplasmic staining while Langerhans' cells and follicular dendritic reticulum cells were unreactive. KP1 recognises a molecule of about 110 kilodaltons in macrophage-rich human tissue when tested by either immunoprecipitation or Western blotting (although the latter procedure also shows two additional components with molecular weights of 70 and 40 kilodaltons). KP1 should be of considerable value for studying disorders of the monocyte/macrophage system, including both reactive and neoplastic states (such as true histiocytic proliferations).

Antibodies, Monoclonal↗
PubMed1988

Use of monoclonal antibody against human neutrophil elastase in normal and leukaemic myeloid cells.

A monoclonal antibody, NP57, was produced and used against the neutrophil granule protein elastase, which selectively stain neutrophils in cryostat and paraffin wax sections. The antibody stains neutrophils and a subpopulation of monocytes in blood smears and neutrophil precursors in bone marrow smears, and gives positive reactions with the cell lines HL60 and U-937. It labelled the blast cells in 68% of cases of acute myeloid leukaemia (M1-M5) but was unreactive with all cases of lymphoid leukaemias. Most of the elastase negative myeloid leukaemias were labelled by monoclonal anti-myeloperoxidase (antibody MPO-7) as were cells from the promyelocytic line HL60. No cases of myeloid leukaemia showed the opposite pattern--that is elastase positive, myeloperoxidase negative, suggesting that the production of myeloperoxidase precedes the onset of elastase synthesis during myeloid maturation. The anti-elastase antibody NP57 is a useful addition to the range of monoclonal antibodies available for the differential diagnosis of acute leukaemia by alkaline phosphatase-antialkaline phosphatase (APAAP) labelling of cell smears; it may also be of value for the histopathological diagnosis of tumour deposits in myeloid leukaemia and for the detection of neutrophils in paraffin sections.

Antibodies, Monoclonal↗
PubMed1988

Protection against complement-mediated cell damage by Ca2+ and Zn2+.

Ca2+ and Zn2+ prevent antibody-dependent complement-induced permeability changes in tonsil lymphocytes and Lettre cells. Lactate dehydrogenase leaks out from Lettre cells at high complement:cell ratios, under which conditions higher concentrations of Ca2+ and Zn2+ are required for protection. Ca2+ and Zn2+ do not inhibit complement activation or C9 binding to Lettre cells, and prevent leakage through preformed lesions. It is concluded that the extent of complement-induced membrane damage depends on the concentration of extracellular Ca2+, and may be modulated by changes in extracellular Ca2+ or Zn2+.

Animals↗
PubMed1986

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations.

Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.

Animals↗
PubMed1985

Isolation of complement-fragment-iC3b-binding proteins by affinity chromatography. The identification of p150,95 as an iC3b-binding protein.

The proteins from labelled human spleen membranes and polymorphonuclear leucocytes which bind to the iC3b fragment of complement component C3 were prepared by iC3b-Sepharose chromatography in the presence of bivalent cations. Complement receptor type 3(CR3) was eluted from iC3b-Sepharose by removal of bivalent cations. Complement receptors type 1 and 2 (present in spleen but not in polymorphonuclear leucocytes) were sequentially eluted by an NaCl gradient. An additional protein of Mr 135 000 was eluted from iC3b-Sepharose under the same conditions as those used to elute CR3. Preabsorption of the starting material on an anti-(CR3 beta-subunit) antibody column before iC3b-Sepharose chromatography removed the alpha- and beta-chains of CR3 and the 135 000-Mr protein. Preabsorption with iC3b-Sepharose before the anti-(CR3 beta-subunit) antibody column showed that iC3b binds CR3 and p150,95, the smallest member of the group of three homologous proteins that share the same beta-subunit.

Antibodies, Monoclonal↗
PubMed1985

Sequential onset of permeability changes in mouse ascites cells induced by Sendai virus.

The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. Depending on virus: cell ratio, Ca2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. A model for the induction of permeability changes is presented.

Animals↗
PubMed1985

Oxonol dyes as monitors of membrane potential: the effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension.

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.

Animals↗
PubMed1985

Permeability changes resulting from virus-cell fusion: temperature-dependence of the contributing processes.

A new assay for membrane fusion, using the fluorescent probe pyrene-sulphonyl-phosphatidyl ethanolamine, has been developed. Fusion between the envelope of Sendai virus and human erythrocytes or Lettre cells has a Q10 of approximately 4 at 37 degrees C, increasing to approximately 7 at 7 degrees C; there is no lag to onset of fusion. Viral neuraminidase has a Q10 of 2.3 between 37 degrees C and 4 degrees C. Its action limits the extent of fusion by causing the elution of virus; this effect is particularly marked at low temperature because of the difference in Q10 for fusion and neuraminidase. The temperature-dependence of the initiation of permeability changes following the removal of inhibitory amounts of Ca2+ is approximately 2; thus membrane fusion is the principal temperature-sensitive step during the permeabilization of cells by Sendai virus. A recovery process, by which cells become insensitive to the removal of Ca2+ and which therefore limits the extent of permeabilization, has a Q10 of 7.4 between 37 degrees C and 21 degrees C. It is concluded that the lag to onset of permeability changes is not due to a lag in virus-cell membrane fusion, but to the gradual acquisition of a threshold level of membrane damage; the extent of permeabilization depends on the rate of fusion relative to the rates of neuraminidase and recovery.

Calcium↗
PubMed1985

Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+.

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.

Animals↗
PubMed1984

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.

Antibodies↗
PubMed1984

Effect of Ca2+-antagonists on virally-induced cell-permeability changes.

Sendai virus-mediated permeability changes in cells are affected by extracellular Ca2+ or Mn2+ as follows: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Drugs that block Ca2+ action in excitable cells, such as verapamil and prenylamine, and drugs that inhibit the action of calmodulin, such as trifluoperazine and R24571, have an effect opposite to that of Ca2+: lag is shortened and extent of leakage is increased. The concentration at which either type of drug shows 50% of maximal effect is similar to the concentration at which 50% of binding by drug to calmodulin is achieved. It is concluded that calmodulin may be involved in protecting cells against virally-mediated membrane damage; alternatively the action of calmodulin-binding drugs may not be as specific as currently thought.

Calcium↗
PubMed1984

The effect of Ca2+ on virus-cell fusion and permeability changes.

Sendai virus-mediated permeability changes in Lettre cells or red blood cells are affected by extracellular Ca2+ in the following way: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Ca2+ neither stimulates nor inhibits fusion of the viral envelope to the plasma membrane of Lettre cells or red blood cells. It is concluded that Ca2+ protects cells against virally-induced permeability changes in a manner not involving membrane fusion.

Calcium↗
PubMed1983

Rat mast cells permeabilized with Sendai virus secrete histamine in response to Ca2+ buffered in the micromolar range.

In the presence of low extracellular Ca2+, Sendai virus generates permeability lesions in the membrane of rat mast cells. This causes leakage of intracellular phosphorylated metabolites and lactate dehydrogenase; it also permits uptake of normally impermeant aqueous solutes such as the complex of Ca2+ with N-hydroxyethylethylenediaminetriacetic acid. We have used this system to buffer the concentration of cytosol Ca2+ in the micromolar range and thus to cause release of the contents of the secretory granules such as histamine and beta-N-acetylglucosaminidase. We argue that such release occurs by a normal exocytotic secretory mechanism for the following reasons. (1) While leakage of cytosol components is progressively inhibited by Ca2+ in the range 1-10 microM, release of histamine is dependent on Ca2+ in this range. Higher concentrations of Ca2+ are inhibitory to both permeabilization and histamine release. (2) While leakage of phosphorylated metabolites is enhanced in metabolically inhibited cells and the leakage of lactate dehydrogenase is insensitive to metabolic inhibition, the release of histamine and beta-N-acetylglucosaminidase is strictly dependent on an intact metabolic process.

Animals↗
PubMed1983

A novel method for measuring intracellular pH and potassium concentration.

The concentration of Na+ and K+ and the pH in the cytoplasm of Lettré cells was measured by monitoring the net flux of H+, Na+, or K+ across the plasma membrane which had been rendered permeable to these ions by the action of Sendai virus. Ion flux was measured directly by analysis of cell composition, or indirectly by observing the change in membrane potential of cells treated with a specific ionophore. Cytoplasmic concentrations of cations were obtained by establishing the concentration of the cation in the medium at which addition of Sendai virus causes no change in cytoplasmic cation content. The value of Lettré-cell pH was confirmed by direct measurement employing 31P nuclear magnetic resonance, and the values of Na+ and K+ concentration were confirmed by analysis of cell cation and water content. Lettré cells suspended at 32 degrees C in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 50 mM Na+ and 80 mM K+.

Animals↗
PubMed1983

Myxoviruses do not induce non-specific alterations in membrane permeability early on in infection.

The permeability characteristics of cells infected with myxoviruses have been studied by measuring the concentrative uptake of nutrients, the concentration of intracellular K+, and the maintenance of the Na+ gradient across the plasma membrane. Cells either show no change at all (Sendai virus-infected BHK cells and measles virus-infected Vero cells) or they show a decreased ability to concentrate nutrients, while intracellular K+ and the Na+ gradient remain unchanged (Sendai and influenza virus-infected L-1210 cells, measles virus-infected lymphocytes and mumps virus-infected L-41 cells). In no case, therefore, was a change observed that resembles the non-specific increase in membrane permeability induced by haemolytic paramyxoviruses (35, 42) or the non-specific membrane leakiness postulated to take place in infected cells (8, 9). A preliminary account of some of these findings has been presented (39).

Animals↗