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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 163 records · Page 9Linked to original sources
PubMed1983

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Amino Acid Sequence↗
PubMed1983

A monoclonal antibody with specificity for murine mu heavy chain which inhibits the formation of antigen-specific direct IgM plaques.

A panel of monoclonal rat antibodies binding to mouse mu heavy chain were tested for their ability to inhibit the formation of antigen-specific plaques in the hemolytic plaque assay. Nine antibodies inhibited SRC-specific direct IgM plaques at high concentrations (greater than 20 micrograms/ml). In contrast to all others, however, one antibody inhibited these plaques at much lower concentrations (down to 0.4 microgram/ml) when added to the assay. This antibody also inhibited plaques formed by cells secreting antibodies against trinitrophenyl or phosphorylcholine determinants. IgG plaques with any of the above specificities were not inhibited. IgM secretion was unaffected by the monoclonal anti-mu antibody. Its inhibitory effect on plaque formation rather appears to be a consequence of its ability to inhibit complement dependent, IgM mediated lysis of erythrocytes. This monoclonal anti-IgM antibody therefore provides a convenient reagent to distinguish specific direct IgM plaques from indirect IgG plaques.

Animals↗
PubMed1983

Requirement for macrophages or for macrophage- or T cell-derived factors in the mitogenic stimulation of murine B lymphocytes by lipopolysaccharides.

Splenic B cells from a variety of mouse strains could be depleted of accessory cells by removal of large cells through velocity sedimentation, followed by adherence to plastic and by passage over Sephadex G-10. Such accessory cell removal abolished the reactivity of the splenic B cells to the mitogen lipopolysaccharide (LPS), as measured by their capacity to polyclonally proliferate and mature to IgM-secreting cells. Accessory cells from different sources, such as peritoneal exudate cells, irradiated spleen cells, cells of the macrophage line P388 D1 and macrophages from a single colony grown from bone marrow precursors in semi-solid media in the presence of colony-stimulating factor all reconstituted LPS reactivity of the accessory cell-depleted B cells. Limiting dilutions of the cells of a single macrophage colony indicated that as little as 30 to 1000 macrophages can reconstitute the polyclonal response of 3 X 10(4) B cells to LPS. Not only activated macrophages, but also activated long-term helper T cell lines and T cell hybridomas, produced supernatant factors which could also restore responsiveness of depleted B cells to LPS.

Animals↗
PubMed1983

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34.

A T cell line was established in long-term tissue culture from spleen cells of BALB/c mice immunized with influenza virus PR8 (A/PR/8/34(H1N1)) by continuous restimulation with PR8 virus and syngeneic x-irradiated spleen cells. This T cell line and clones derived from it have now been propagated for over 1 yr "in vitro". The T cell line and the clones, upon stimulation with Con A in the absence of antigen and irradiated spleen cells, as upon stimulation in the presence of antigen and adherent cells, produce T cell growth factors, B cell replication and maturation factors, and colony-stimulating factors, as tested by restimulation of proliferation of a clone of cytolytic murine T cells, of lipopolysaccharide-activated B cell blasts, and by macrophage/granulocyte colony formation of murine bone marrow cells, respectively. The T cell line and the clones help syngeneic B cells in the presence of syngeneic irradiated spleen cells and PR8 virus to proliferate and mature into clones of cells secreting virus-specific antibodies. In the presence of PR8 virus and of a bystander antigen, such as sheep red cells, B cells specific for this bystander antigen are also induced. Adoptive transfer of the T cells i.v. into syngeneic nu/nu BALB/c mice enable the recipient mice to produce, upon challenge with PR8, virus-specific antibodies and to clear virus infection of the lung. The virus-specific T cell line and the clones are restricted by H-2 antigens of the d-haplotype, probably by Iad, and recognize a determinant on the hemagglutinin (HA) molecule of PR8 virus. Fifty to 100 fmol of virus-associated or free HA suffice for stimulation as measured by proliferation assays. The stimulating determinant is present on the HA of all natural virus isolates of the H1 subtype, is absent from virus isolates of the H2 and H3 subtypes, and is abolished if glutamic acid at position 115 of the HA1 polypeptide of PR8 is replaced by lysine.

Animals↗
PubMed1982

How to produce antigen-specific antibodies in tissue culture.

Before antigen ever enters the immune system we believe that lymphocytes with antigen-specific receptors preexist. For B lymphocytes we know that these antigen binding structures are immunoglobulin (Ig) molecules, antibodies. One B cell makes one of the many Ig molecules and displays them in many copies on the surface membrane. From the observed extreme diversity of antibody molecules we are tempted to think that any antigen can find its specific binding receptors, i.e., its preexisting antibody molecules. Essential problems of how to produce large quantities of such antigen-specific antibodies are how to stimulate the preexisting lymphocytes from their resting state and how to increase the production of that specific antibody. Proliferation and maturation to high rate-antibody secreting cells follows stimulation and therefore provides ways by which the synthesis of the preexisting antibodies is expanded. The motivation to understand the induction of specific antibody production and secretion in the corresponding B lymphocytes in vitro may come either from the desire to understand the rules that govern the reactions of B cells leading to such antibody production, or from the need to do it outside of the body due to the potentially harmful effects of some antigens in vivo.

Animals↗
PubMed1981

T cell-dependent activation of resting B cells: requirement for both nonspecific unrestricted and antigen-specific Ia-restricted soluble factors.

Cloned murine helper T cells, restricted to the Iab antigens of the major histocompatibility locus and specific for horse erythrocytes as a foreign antigen, produce, in cooperation with antigen and histocompatible adherent cells, soluble factors that replace the helper T cells in their action on B cells. Three types of factors can be distinguished on the basis of molecular weight: proteins having apparent Mr 30,000 (p30) that act antigen- and Ia-nonspecifically as replication- and maturation-inducing factors and proteins having apparent Mr 55,000 (p55) and 125,000 (p125) that act on resting B cells in an Ia-specific, restricted fashion. Neither horse erythrocytes (a T-cell specific antigen) nor p55 and p125, alone or together, stimulate resting B cells to proliferation and maturation. Double occupancy by antigen and p55 or p125, however, renders Ia-compatible, but not Ia-incompatible, resting B cells susceptible to stimulation. The subsequent addition of p30 to these "excited" B cells then results in the proliferation and maturation of clones of horse erythrocyte-specific resting B cells, which then replicate under the stimulatory action of p30. p30 do not bind antigen, nor do they bind anti-Ia or anti-immunoglobulin antibodies. p55 are bound by anti-heavy chain variable region antibodies. p55 are bound by anti-heavy chain variable region antibodies, but not by anti-heavy or anti-light chain constant region antibodies or anti-Ia antibodies. p125 molecules bind horse but not sheep erythrocytes and are bound by anti-heavy chain variable region, but not by anti-heavy or light chain constant region or anti-Ia antibodies. p55 and p125 are likely to be soluble analogues of the antigen-specific, Ia-restricted T-cell receptors of the cloned helper T cells.

Animals↗
PubMed1981

Cellular interaction and the environment in lymphocyte development: the roles of antigen, histocompatibility, and growth factors in T cell-dependent B cell stimulation.

T cell-dependent B cell stimulation depends on cellular interactions between macrophages, T cells, and B cells. T cells recognize antigen in the context of Ia determinants on macrophages. This leads to the activation of helper T cells and to the production of helper factor(s) either by the activated helper T cells or by the macrophages. Cloned lines of helper T cells produce factors("help') for B cell replication and Ig secretion in the presence of histocompatible macrophages and of specific antigen. These factors stimulate histocompatible, as well as histoincompatible, mitogen-activated B cell blasts polyclonally. Thus, neither antigen nor histocompatibility, but antigen-unspecific, soluble factors are required to stimulate an activated B cell blast through the next round of division. Small resting B cells, however, are not stimulated to replication by these factors, but only to polyclonal, H-2-unrestricted maturation to Ig-secreting cells. Replication (and Ig secretion) of small resting B cells, however, is only induced when antigen-specific small B cells bind their specific antigen via surface Ig molecules and interact with histocompatible helper T cells. The preference of the resting B cells for such collaboration with T cell help is mapped to the K-end of the H-2 locus and probably constitutes the antigen expressed on B cells by the I region. It appears that a resting B cell is excited by the binding of specific antigen to surface Ig and by the interaction of its surface Ia with helper T cells. After this dual recognition the excited B cell can be stimulated by the antigen-unspecific factor(s) generated by the interaction of helper T cells, adherent cells and antigen to initiate growth. Immune induction of a B cell thus involves three controlling elements: Ig, Ia, and B cell growth factor receptors.

Animals↗
PubMed1980

Antigen-specific T-helper cells stimulate H-2-compatible and H-2-incompatible B-cell blasts polyclonally.

Lipopolysaccharide (LPS)-activated B-cell blasts from C57BL/6J nu/nu spleen cells develop into IgM-secreting clones after stimulation by antigen-specific T-helper cells of C57BL/6J origin. Although induction of help is antigen-dependent, help itself acts polyclonally. 1 of 1--3 B-cell blasts is restimulated in a homologous fashion by LPS, or in a heterologous fashion by sheep erythrocyte (SRC)- or horse erythrocyte (HRC)-activated T-helper cells. The repertoire of activated B-cell blasts reflects the polyclonal nature of activation: approximately 1 in 1,000--3,000 restimulated B-cell blasts is specific for SRC, 1 in 300--1,000 is specific for HRC, and 1 in 100--300 specific for trinitrophenylated SRC (TNP30-SRC). B-cell blasts that are either H-2 compatible or H-2 incompatible with the antigen-activated T-cell help are stimulated polyclonally in similar high frequencies. Thus, neither antigen nor H-2 compatibility are required to stimulate a B-cell blast into the next cell cycle.

Animals↗
PubMed1980

Murine embryonic blood between day 10 and 13 of gestation as a source of immature precursor B cells.

Pre-B cells which show a delayed development of responsiveness to the B cell mitogen lipopolysaccharide (LPS) are found in murine embryonic blood between day 10 and 14 of gestation. The highest frequency of pre-B cells in embryonic blood is found at day 12 of gestation. All pre-B cells of embryonic blood, isolated at different days of gestation, develop responsiveness in vitro to LPS at the same time, i.e. around day 14 of gestation, yielding a peak of LPS-induced IgM plaque-forming cells at the equivalent of day 19 of gestation. Dextran sulfate supports the development of pre-B cells into LPS-reactive B cells; by itself, however, it does not stimulate these B cells into IgM plaque-forming cells. The wave of pre-B cells in embryonic blood occurs parallel in time to that found previously in placenta. Fetal liver and yolk sac, at this time of embryonic development, do not contain significant numbers of pre-B cells, although they are connected with the placenta and in contact with embryonic blood by circulation.

Animals↗
PubMed1980

T-cell-dependent B-cell stimulation is H-2 restricted and antigen dependent only at the resting B-cell level.

Cloned lines of helper thymus-derived (T) cells produce help for bone marrow-derived (B) cell growth and Ig secretion in the presence of histocompatible adherent cells and of specific antigen. This help stimulates histocompatible as well as histoincompatible B-cell blasts polyclonally. Thus, neither antigen nor histocompatibility, but antigen-unspecific factor(s) for growth and Ig secretion are required to stimulate a B-cell blast through the next round of division. On the other hand, only histocompatible, resting, small B cells, and only those binding their specific antigen, can be stimulated by antigen-activated T-cell help to initiate growth and Ig secretion. The preference of the resting B cells for such collaboration with T-cell help is mapped to the K end of the H-2 histocompatibility locus, and probably constitutes the antigen expressed on B cells by the immune response (I) region. It appears that a resting B cell is excited by the binding of specific antigen to surface Ig and by the interaction of its surface Ia antigen with helper T cells. After this dual recognition the excited B cell can be stimulated by the antigen-unspecific factor(s) generated by the interaction of helper T-cells, adherent cells, and antigen to initiate replication.

Animals↗