Biomedical subjects

M D Boyle

Publications and source records attributed to M D Boyle.

At least 91 records · Page 5Linked to original sources

PubMed1988

Identification of protein A from Staphylococcus intermedius isolated from canine skin.

Protein A was identified in cell wall-bound and secreted forms from Staphylococcus intermedius isolated from canine skin. A direct binding radioimmunoassay for the detection of bacterial surface Fc receptors identified 48 of 50 S intermedius isolates that contained cell wall-bound protein A. Using a competitive binding radioimmunoassay for the detection of Fc-reactive proteins in bacterial culture supernatants, we identified 9 of 50 clinical isolates of S intermedius that secreted measurable quantities of an Fc receptor into the culture medium. Concentrated culture supernatants from these isolates were analyzed by western blotting techniques and probed with either a radiolabeled human IgG Fc-specific probe or a radiolabeled affinity-purified chicken antibody against protein A. The studies reported here confirmed that Fc receptors are secreted by S intermedius isolates from dogs and are antigenically and functionally similar or are identical to staphylococcal protein A. Analysis of Fc receptor secretion by S intermedius strains, isolated from dogs with a variety of dermatologic conditions, suggested a trend between severity of skin disease and the extent of Fc receptor secretion.

Animals↗

PubMed1987

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo.

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.

Animals↗

PubMed1987

Inactivation of the proteolytic activity of mouse nerve growth factor by human C1(activated)-inhibitor.

The interaction between the serine protease gamma subunit of NGF (gamma-NGF) and human C1(activated)-inhibitor (C1-Inh) has been studied. C1-Inh inactivates the protease activity of gamma-NGF as measured by its ability to cleave the synthetic substrate benzoyl-arginine-p-nitroanilide (L-BAPNA). Experiments in which gamma-NGF and C1-Inh were mixed at differing molar ratios indicated that inhibition was due to the formation of a 1:1 stoichiometric complex. Analysis of the interaction of 125I-labeled gamma-NGF with C1-Inh by SDS-PAGE and autoradiography indicated that a covalent bond was formed between gamma-NGF and C1-Inh. The covalent bond was hydrolyzed by hydroxylamine, which suggested that the two proteins were linked via an acyl linkage. The formation of this complex was time dependent and required the proteolytic activity of the gamma-NGF.

Animals↗

PubMed1987

Regulation of the activation of C1 in serum.

C1 has been partially purified from serum and its ability to activate has been studied. The activation status of C1 was measured using a sensitive hemolytic assay which allowed the activation status of C1 in serum to be monitored. C1 did not activate in serum but would spontaneously activate when separated from certain other serum proteins, in particular, the C1-inhibitor protein. The activation of C1 was time and concn dependent, but addition of fully activated C1 did not affect either the rate or extent of activation. The activation of C1 could be inhibited reversibly by C1-inhibitor. This action of the C1-inhibitor was over and above its ability to regulate fully activated C1 by covalent bond formation with either the serine esterase of C1s or C1r. The results presented suggest that the C1-inhibitor plays a dual role in the regulation of C1 activation. The regulatory actions of C1-inhibitor would account for the presence of C1 in a zymogen form and for the episodic nature of complement consumption observed in individuals with genetic deficiencies of the C1-inhibitor protein.

Chromatography, Ion Exchange↗

PubMed1987

Selective loss of expression of a tumor-associated antigen on a human leukemia cell line induced by treatment with monoclonal antibody and complement.

The sensitivity of a common acute lymphoblastic leukemia-associated antigen (cALLa)-positive, human leukemia pre-B-cell line to killing by antibody and complement was studied. A stable subpopulation was selected by its ability to survive four sequential treatments with excess monoclonal antibody (MoAb) directed against an Mr 24,000 glycoprotein associated with human leukemia cells and excess rabbit complement. Analysis of the antigen expression by individual cells within the parental and the selected cell populations was achieved by flow cytometry and demonstrated a marked decrease of the leukemia-associated antigen expression on individual cells within the selected subpopulation. These low-antigen-density cells were stable in subculture, and the immunoglobulin heavy-chain gene rearrangement of the parent population and the low-antigen-density subpopulation were identical, indicating that they were derived from a single cell source. The selection of this subpopulation was specific in that the expression of a second antigen recognized by a cALLa-specific MoAb was not affected. The presence of subpopulations of tumor cells with low levels of surface antigen expression that are resistant to killing upon addition of excess antibody and complement will prove to be an obstacle to the use of this approach to eliminate tumor cells from bone marrow that is to be used for autologous transplantation.

Antibodies, Monoclonal↗

PubMed1987

Identification of a specific receptor for plasmin on a group A streptococcus.

Certain group A streptococci demonstrate surface receptors that bind selectively to the key fibrinolytic enzyme, plasmin. These bacteria show no reactivity with the zymogen protein plasminogen or with other serine class proteases, such as trypsin or urokinase. Bacterium-bound plasmin retains its ability to cleave synthetic substrates and its ability to hydrolyze a fibrin clot. The bacterium-bound plasmin is not effectively regulated by its physiological regulator, alpha 2-plasmin inhibitor. This study is the first report of a bacterium-associated receptor for plasmin.

Fibrinolysin↗

PubMed1987

Low antigen density tumor cells: an obstacle to effective autologous bone marrow purging?

Autologous bone marrow transplantation is a potentially curative approach to the treatment of various tumors that are refractory to conventional therapies. A major problem associated with the procedure is the possibility of tumor cell contamination in the autologous graft used to reconstitute the patient's immune system after supralethal chemoradiotherapy. A variety of different approaches to eliminating tumor cells from bone marrow have been proposed and tested. These include destruction of tumor cells with antibody and complement, use of antibody conjugated to drugs or toxins, and the physical separation of antibody-coated tumor cells by attachment to magnetic microspheres. Each of these approaches has different limitations and technical problems. One problem common to all, however, is that the tumor cells most likely to avoid removal are those demonstrating a low level of surface antigen. In this paper we have offered a practical approach to amplifying the unique surface antigen expression in order to enable this elusive tumor cell population to be eliminated. The approach proposed is adaptable to all the techniques currently being studied, since it is designed to add additional antigens to tumor cells which can then be used as targets at which to direct the various purging strategies.

Antibodies, Monoclonal↗

PubMed1986

Identification of a unique receptor on a group A streptococcus for the Fc region of human IgG3.

Receptors that bind to the Fc region of all four human IgG subclasses have been described on a number of strains of group A streptococci. In this study, we have demonstrated that these immunoglobulin binding properties are mediated by two distinct Fc receptors. The first receptor, with a Mr of approximately 56,000, binds to human IgG1, IgG2, and IgG4, but not to IgG3. A second receptor, with a Mr of approximately 38,000, binds exclusively to human immunoglobulins of the IgG3 subclass.

Animals↗

PubMed1986

Influence of dipeptides on the interaction of immunoglobulins with bacterial Fc receptors.

Binding of radiolabeled human IgG to bacteria expressing type I, type II, or type III Fc receptors in the presence of glycyl-glycine, glycyl-tyrosine, glycyl-histidine, glycyl-leucine, or glycyl-phenylalanine was studied. No inhibition of labeled human IgG binding to type I or type III Fc receptor positive bacteria was observed by any of the dipeptides. Inhibition of binding of labeled human IgG1, IgG2, and IgG4, but not IgG3, indicated the presence of two distinct Fc receptors associated with the type II Fc receptor-positive group A streptococcal strain.

Dipeptides↗

PubMed1986

Isolation and partial characterization of a type II Fc receptor from a group A streptococcus.

A group A streptococcal strain rich in Fc receptors was selected by an immunoblotting technique and used as the source for isolation of a functionally active Fc receptor. A variety of extraction techniques were compared including (1) heat extraction at neutral, acid or alkaline pH, (2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin, papain or phage lysin, or (3) autoclaving or heating in the presence of sodium dodecyl sulfate. The most homogeneous receptor was recovered following heat extraction and contained two molecular weight forms. The major form had a molecular weight of 56 000 daltons and the minor form had a molecular weight of 38 000 daltons. These two proteins could be isolated without loss of activity by binding to and elution from a column of immobilized human IgG. An antibody prepared against a single form of the affinity purified receptor demonstrated reactivity with both molecular weight forms of the heat extracted receptor. The group A receptor was found to be both antigenically and physicochemically distinct from either the type I receptor found on the majority of Staphylococcus aureus strains or the type III Fc receptors found on the majority of group C streptococcal strains.

Binding, Competitive↗

PubMed1986

Comparison of type III Fc receptors associated with group C and group G streptococci.

The type III Fc receptors present on or secreted by a series of group C and G streptococcal strains were studied. All strains capable of binding radiolabeled human IgG were shown to do so via an antigenically related Fc receptor. Treatment of any of the bacterial strains with papain or trypsin resulted in solubilization of Fc receptor activity. The pattern of Fc receptor activity recovered following enzyme treatment was not uniform. Differences were observed both between group C and G strains as well as within group C and G strains. Analysis of secreted Fc receptors indicated the presence of five molecular forms of Fc receptor. Each form was present at some level in the supernatant of every group C and G strain studied. The relative concn of each form of receptor secreted varied from strain to strain. The Fc receptor activity secreted by each strain demonstrated a similar affinity for the Fc region of human IgG and all were antigenically related. These results suggest that there is a family of closely related Fc receptors associated with group C and G streptococcal strains.

Binding, Competitive↗

PubMed1986

Restriction in the lytic efficiency of complement of different erythrocyte targets: a re-examination of the activities of horse C8 and C9.

Differences in the lytic efficiency of different complement sources have frequently been observed. This effect has been shown to be related to both the species of the target erythrocyte and the species composition of terminal complement components within the 5b-9 membrane attack complex. The majority of studies have indicated that the source of C9 is critical in controlling the range of erythrocyte species that can be lysed efficiently. One exception to this general finding was the report by Lachmann et al., 1973 (Immunology 24, 135-145), using horse serum as a complement source. In that study, horse C8 rather than C9 was implicated as the critical component. In this study, we have re-examined this observation and find that the restricted hemolytic potential of horse complement correlates absolutely with the presence of horse C9. The reason for the differences between our findings and those of the earlier study are discussed.

Animals↗

PubMed1986

Production of Brucella abortus-specific protein A-reactive antibodies (IgG2) in infected and vaccinated cattle.

The IgG2 anti-Brucella antibody response of cattle to Brucella vaccination and infection was measured. Three groups of animals were studied; Group 1 contained 11 non-vaccinated cows, Group 2, 17 cows vaccinated with a low dose of Strain 19 vaccine and Group 3, 17 cows vaccinated with a high dose of Strain 19 vaccine. All animals were challenged at Week 33 with an infectious isolate of B. abortus (Strain 2308). Studies of the IgG2 antibodies response indicated an absolute correlation between anti-Brucella IgG2 levels and infection of the animal. All animals showing reciprocal titers of greater than or equal to 3000 (16 of 45 tested) were found to be positive for the challenge organism at slaughter. Animals with reciprocal IgG2 titers less than or equal to 1000 (29 of 45 tested) were found to be negative for the challenge organism at the time of slaughter. The predictive value of IgG2 antibody levels for infection held for animals in all three groups and consequently this suggests that monitoring of specific IgG2 anti-Brucella antibody levels may be of value in detection of Brucella-infected cattle.

Animals↗

PubMed1986

Analysis of surface receptor expression on bacteria isolated from patients with endocarditis.

Fifteen bacterial isolates (12 streptococcal and 3 staphylococcal strains) from patients with bacterial endocarditis were screened for a variety of surface receptors, in an attempt to identify a common feature that might contribute to their ability to attach to and colonize damaged heart tissue. The bacterial receptors screened for, using a dot-blot autoradiographic procedure, included those for the Fc region of human IgG, fibrinogen, fibronectin and human C1q. Bacteria with receptors for each of the probes used could be identified, but no common receptor was present on all endocarditis-causing strains.

Binding Sites↗

PubMed1986

Isolation and characterization of type IIa and type IIb Fc receptors from a group A streptococcus.

Certain group A streptococcal strains have been reported to express two distinct type II receptors that bind to the Fc region of human IgG. In this study, we have isolated and characterized these two type II Fc receptors and characterized their reactivity with differing species of IgG. The type IIa receptor was found to be a 56,000 molecular weight protein which binds human IgG1, IgG2 and IgG4, in addition to pig and rabbit IgG. The type IIb receptor was found to be a 38,000 molecular weight protein that bound exclusively to human IgG3. Neither the type IIa nor the type IIb receptor bound to goat, cow, dog, rat, or sheep IgG. Monospecific polyclonal antibodies were prepared against both the type IIa and type IIb Fc receptors. These antibodies demonstrated that the type IIa and type IIb were antigenically closely related and could not be distinguished from each other on the basis of their reactivity with either antibody. The distribution of type IIa and type IIb Fc receptors on a variety of different nephritogenic and non-nephritogenic group A streptococcal strains was documented.

Chromatography, Affinity↗

PubMed1985

Nerve growth factor. A structural relationship between its proteolytic and leukocyte-chemotactic active sites.

High molecular weight mouse nerve growth factor (HMW-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of HMW-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function-proteolysis as well as nonproteolytically mediated chemotaxis.

Animals↗

PubMed1985

Nerve growth factor accelerates the early cellular events associated with wound healing.

The effects of injecting nerve growth factor (NGF) into subdermal air sacs on the backs of mice was studied. Sequential infiltration of cells into the lining of the sacs was observed. The initial cell type to infiltrate was the polymorphonuclear leukocyte, followed by highly vacuolated mononuclear cells, and then by fibroblast-like cells. This resembles the classical pattern of cellular responses during the normal process of wound healing. The kinetics for the peak accumulation of each cell type were dependent upon the concentration of NGF injected, and significant acceleration of infiltration of each cell type was observed with as little as 1 nM NGF. A similar acceleration of cellular infiltration was observed when mice were injected with the synthetic chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine. This finding suggests that application of any chemotactic factor to wound sites may lead to accelerated tissue repair and an enhanced rate of wound contraction. The observation that NGF can act as a chemotactic factor in vivo may explain its ability to accelerate the healing of experimentally induced wounds in mice.

Animals↗

PubMed1985

A rapid alternative to the clonogenic assay for measuring antibody and complement-mediated killing of tumor cells.

A study of the methods used to quantitate killing of tumor cells by antibody and complement has highlighted a number of problems. Using leukemia as a model we have found that the release of 51Cr from labeled tumor cells treated with antibody and complement can be an equivocal measure of cell viability. Combined with its restricted sensitivity (less than a 2 log range of cell killing) this makes this widely used assay of questionable value for detecting small numbers of viable cells, or for identifying subpopulations of complement-resistant cells. As an alternative we have developed a [125I]iododeoxyuridine (125IUdR) uptake assay, that combines the simplicity and rapidity of the 51Cr release technique with the sensitivity of a clonogenic assay. This method eliminates the problem of spontaneous isotope release, inherent in prelabeling assays, and variability from experiment to experiment can be avoided by including a viable cell standard curve within each assay. The sensitivity of the 125IUdR uptake method, which can be completed within a day, is similar to that of a 10 day methylcellulose cloning assay and was capable of detecting the presence of a minor subpopulation of complement-resistant tumor cells.

Animals↗