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D H Conrad

Publications and source records attributed to D H Conrad.

107 records · Page 6Linked to original sources
PubMed1979

Lentil-lectin affinity chromatography of surface glycoproteins and the receptor for IgE from rat basophilic leukemia cells.

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).

Animals↗
PubMed1979

Complement receptor binding of C3b-coated cells treated with C3b inactivator, beta 1H globulin and trypsin.

Treatment of 125I-C3b bound to EAC1423b with C3b inactivator (C3bINA) and beta 1H globulin (beta 1H) cleaved the alpha-chain of C3b into 65,000- and 42,000-dalton fragments, both of which remained disulfide-bonded to the intact beta-chain (C3bi). Subsequent treatment with trypsin (0.1 microgram/ml) released 125I into the supernatant and yielded cells coated with a 33,000-dalton fragment of alpha-chain, presumably C3d. These results are in agreement with those obtained by others using fluid phase C3b. C3b-coated cells (EAC1423b) adhered to complement (C) receptors on human erythrocytes, glomeruli, and monocytes. C3bi-coated cells adhered to the receptors on glomeruli and monocytes, but not to those on human erythrocytes. C3d-coated cells adhered only to the monocyte receptors. The findings suggest that the glomerular C receptor recognizes portions of the C3 molecule different from those recognized by either the erythrocyte or monocyte receptors.

Animals↗
PubMed1978

Interaction of beta1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding.

Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.

Animals↗
PubMed1978

A new concept of triggering mechanisms of IgE-mediated histamine release.

It is generally accepted that an initial step of reaginic hypersensitivity reactions is a bridging of mast cell--bound IgE antibody molecules by antigen. Since IgE molecules are firmly bound to receptors on mast cells, bridging of cell-bound IgE molecules probably brings receptor molecules into close proximity. A hypothesis was therefore presented that such a local change in membrane structure and/or possible interaction between adjacent receptor molecules may be triggering mechanisms of IgE-mediated histamine release. The hypothesis was tested by use of antibodies against "exposed portion" of receptor molecules on rat basophilic leukemia cells. It was found that antireceptor antibodies and its F(ab')2fragments induced noncytotoxic histamine release from normal rat mast cells without participation of IgE, while the monovalent Fab' fragments of the antibody failed to do so. However, sensitization of normal rat skin with the Fab' fragments followed by an intravenous injection of antirabbit IgG induced skin reactions. These findings support the concept that bridging of receptors rather than polymerization of IgE molecules is responsible for the activation of membrane-associated enzymes which in turn leads to histamine release.

Animals↗
PubMed1977

Murine and rat IgE: relationships in terms of binding to cell receptors and to antibodies against rat epsilon chain.

The similarity between murine and rat IgE was examined in terms of their fixation to target cells and interaction with monospecific antibodies to rat epsilon-chain (anti-epsilon). Purified rat monoclonal IgE (IgEr) was found to block the fixation of murine reagin (IgEm) to mouse and rat skin and to rat basophilic leukemia (RBL) cells. The capacities of mouse reaginic serum (MRS), rat reaginic serum, and IgEr to inhibit the binding of radiolabeled 125I-IgEr to RBL cells were shown to be similar. These results suggest that the binding of IgE of either species occurs on the same or on adjacent receptor sites of mast cells and RBL cells. The antigenic cross-reactivity between IgEm and IgEr was established by depletion of the reaginic activity from MRS by treatment of MRS with anti-epsilon. The reaginic activity of MRS could be recovered by the addition of IgEr to anti-epsilon:IgEm complexes. From these findings it may be inferred that i) IgEm and IgEr share some antigenic determinants and ii) the regions of the immunoglobulins responsible for fixation to receptors on mast cells and RBL cells are identical or similar.

Animals↗
PubMed1976

Characterization of the target cell receptor for IgE. II. Polyacrylamide gel analysis of the surface IgE receptor from normal rat mast cells and from rat basophilic leukemia cells.

Purified rat peritoneal mast cells (RMC) and cultured rat basophilic leukemia (RBL) cells were surface labeled with 125I by using lactoperoxidase, incubated with unlabeled rat monoclonal IgE and subjected to solubilization by treatment with Nonidet P-40 (NP-40). With both cell types significant amounts of radioiodinated material could be specifically precipitated by a "sandwich" system consisting of rabbit anti-rat epsilon-chain and goat anti-rabbit Ig. The precipitates were dissociated with sodium dodecyl sulfate (SDS) and urea and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. With RMC three radioactive bands were seen. One corresponded to IgE present on the RMC at the time of isolation. A small band migrating in the region of light chain was seen with both sepcific (anti-IgE) and control precipitates. It showed no demonstrable relationship to IgE. The major radioactive band corresponded to a m.w. of 62,000. This band was dependent upon the presence of IgE and was not found when non-IgE binding control cells were used. With RBL cells, only the IgE-dependent 62,000 dalton peak was present. Saturation of the IgE receptor sites of the RMC or RBL cells before lactoperoxidase labeling almost totally eliminated this radioactive band, indicating that cell-bound IgE rendered this membrane component inaccessible to the radiolabel. These results strongly suggest that this cellular component is identical, at least in part, with the target cell surface receptor for reaginic antibody. The data also further support the hypothesis that the neoplastic RBL cells have a normal surface receptor for IgE.

Animals↗
PubMed1975

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.

Animals↗
PubMed1975

Binding parameters of the interaction between rat IgE and rat mast cell receptors.

Rat monoclonal IgE was purified by a combination of gel filtration and isoelectric focusing. The binding parameters of the IgE-target cell interaction were studied. The target cells used were normal rat peritoneal mast cells (RMC) and neoplastic rat basophilic leukocytes (RBL). BINDING assays were performed by adding different amounts of IgE to a constant cell number. A Scatchard analysis of the data indicated that both cell types have a similar affinity for rat IgE; the equilibrium constant was in the order of 10-9 M-minus 1. However, RBL have a higher number of receptor sites per cell--approximately 6 times 10-5 versus 3 times 10-5 for normal RMC. Since RMC have more than double the surface area of RBL, the data indicate a much higher receptor density on RBL.

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PubMedDate not supplied

IL-4 production by T depleted cells from Nippostrongylus brasiliensis infected mice.

Interleukin-4 (IL-4) is known to be involved in both the in vivo IgE response and the elevated B cell IgE Fc receptor (Fc epsilon R11) expression seen after a parasite infection. To further analyze the relationship between Fc epsilon R11 expression and IL-4 production, purified B cells from uninfected, Nippostrongylus brasiliensis (Nbr) infected and from goat anti-mouse IgD (GaM delta) injected mice were isolated on various days post-treatment. The Fc episolon R11 levels on purified B cells from normal mice decreased after an overnight culture in media alone and addition of IL-4 to these cultures resulted in a 4 to 13-fold enhancement of Fc epsilon R11 levels. In contrast, the Fc epsilon R11 levels on B cells from Nbr infected mice were elevated after an overnight culture in media alone and addition of IL-4 did not further enhance the already upregulated Fc epsilon R11 levels. Overnight culture of purified B cell blasts from Nbr infected mice in the presence of an anti-IL-4 monoclonal antibody (11B11) caused the elevated Fc epsilon R11 levels to return to levels seen in normal mice, without affecting the Fc epsilon R11 levels on purified Go or B cell blasts from uninfected mice or Go B cells from Nbr infected mice. 11B11 also inhibited the elevated Fc epsilon R11 levels on highly purified B cells obtained by FACS sorting the non-adherent spleen cell population for class II+ cells. In contrast to Nbr infection, the Fc epsilon R11 levels on B cells were downregulated in the GaM delta injected mice. However, analogous to the Nbr system, the Fc epsilon R11 levels were unresponsive to the addition of exogenous IL-4. This study indicates that IL-4 production is seen in T depleted splenocytes and that this alternate source of IL-4 serves to maintain the elevated Fc epsilon R11 levels on B cells.

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