Biomedical subjects

A H Sehon

Publications and source records attributed to A H Sehon.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.

Animals↗

PubMed1992

Mapping of antibody binding epitopes of a recombinant Poa p IX allergen.

Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures. The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E. coli with aid of the plasmid expression vector, pWR590.1. The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies. It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3. Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein. The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s). Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.

Allergens↗

PubMed1992

Suppression of antibody responses in rats to murine anti-CD4 monoclonal antibodies by conjugates with monomethoxypolyethylene glycol.

The effectiveness of therapeutically relevant xenogeneic monoclonal antibodies (MoAb) may be counteracted by their inherent immunogenicity. Since conjugates of diverse proteins with mono-methoxypolyethylene glycol (mPEG) were shown to induce Ag-specific tolerance in mice and rats, we used outbred rats in this study as an experimental model for establishing the tolerogenicity of mPEG conjugates of murine MoAb. The results demonstrate that: (i) murine anti-rat CD4 MoAb (W3/25) were more immunogenic than murine anti-human CD4 MoAb (MAX.16H5) in rats; (ii) W3/25 preferentially induced an anti-idiotypic (anti-id) antibody response; and (iii) antibodies to both common and idiotypic determinants could be suppressed in rats by treatment with W3/25(mPEG)28.

Animals↗

PubMed1992

Neuro-hormonal host defence in endotoxin shock.

The sensitivity (LD100) of mice to lipopolysaccharide (LPS) endotoxin and to its toxic moiety, lipid A (LA), increased 500-fold after adrenalectomy (ADX). Inhibition of glucocorticoid synthesis in intact mice by metyrapone had a similar, though less dramatic, sensitizing effect to LPS. In ADX mice, the serum level of tumor necrosis factor-alpha (TNF) was 40-60 times higher than that in controls at 2 h after LPS/LA treatment. In intact mice the serum corticosterone level fell 1 h after lipid A injection to below detectable levels, which was followed by a brisk increase reaching the peak level of 48-50 micrograms/100 ml at 2 h. Both TNF production and the lethal effect of PLS/LA could be inhibited in ADX mice by glucocorticoid treatment. Plasma prolactin was increased significantly 1 h after endotoxin administration in both intact and ADX animals.

Adrenalectomy↗

PubMed1991

Downregulation of helper T cells by an antigen-specific monoclonal Ts factor.

The findings of previous studies in this laboratory demonstrating that conjugates of human monoclonal (myeloma) IgG (HIgG) and monomethoxypolyethylene glycol (mPEG) were able to induce in mice antigen-specific tolerance and CD8+ suppressor T (Ts) cells were confirmed in the present study. An extract (TsF) of a nonhybridized clone of Ts cells (viz., clone 23.32), which had been derived from spleen cells of mice tolerized with HIgG(mPEG)26, was shown to possess antigen-specific suppressive activity. This monoclonal TsF was able to specifically suppress in vitro antibody formation only if it was present from the beginning of the culture. From the results of the cellular dissection of the system used it was concluded that (i) the TsF had no effect on fully differentiated primed B cells or plasma cells, and (ii) the TsF inactivated carrier-primed Th cells when the culture contained concomitantly naive CD8+ T cells, accessory cells, and antigen. These data support the view that the monoclonal TsF exerted its downregulating effect on Th cells only if it could first interact with a CD8+ T cell, in the presence of accessory cells and antigen.

Animals↗

PubMed1991

Development and characterization of ouabain-resistant human fusion partners.

The ouabain-resistant mutant cell lines, HOA-1 and HOA-20 were developed from WI-L2-729-HF2 by cloning with increasing concentration of ouabain. Both parent and mutant cell lines were resistant to base analogues, 6-thioguanine (6-TG) and 8-azaguanine (8-AG) to the level of 20 micrograms/ml in the culture medium. The parent cell line WI-L2-729-HF2 was highly sensitive to ouabain, whereas HOA-1 and HOA-20 were resistant to ouabain to the level of 1 microM and 20 microM, respectively. However, all the cell lines were sensitive to HAT-selective medium which is essential for hybrid selection after fusion. All three lymphoblastoid cell lines were positive for Epstein-Barr virus nuclear antigen (EBNA), secreted TNF-beta (lymphotoxin) without any external stimulation, secreted trace amounts of IgG(kappa), which was also present in their cytoplasm and had IgM(kappa) as surface bound immunoglobulin. They also expressed the CD20, CD71 (transferrin receptor) as surface antigens. In addition to these antigens, HOA-20 also expressed CD38 antigen. The karyotype analysis of these cell lines revealed modal chromosomal numbers ranging from 40 to 47. The HLA-A, -B and -C antigens expressed by WI-L2-729-HF2 and its mutants HOA-1 and HOA-20 were identical. Both the HOA-1 and HOA-20 mutants were found suitable for the generation of hybrids after fusion with EBV-transformed human B-lymphocytes.

Antibodies, Monoclonal↗

PubMed1991

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen.

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.

Allergens↗

PubMed1991

Suppressor T cells induced in vivo by tolerogenic conjugates of a given antigen and monomethoxypolyethylene glycol downregulate antibody formation also to a second antigen, if the latter is presented as a covalent adduct with the former.

Recent results of studies employing tolerogenic monomethoxypolyethylene glycol (mPEG) conjugates of antigens are briefly reviewed. Administration of antigen (mPEG)n conjugates into mice induced antigen-specific suppressor T (Ts) cells, from which a suppressor factor (TsF) was extracted. These Ts cells were cloned and shown to be Thy1.2+, CD3+, CD4-, CD5-, CD8+ and to express the alpha beta heterodimer of conventional T cell receptors (TCR). The TsF of a clone of OVA-specific Ts cells shared the epitopes of the alpha and beta chains of TCR, whereas the TsF of T cells of an HIgG-specific clone shared only the epitope of the alpha chains of TCR; OVA and HIgG represent ovalbumin and human monoclonal (myeloma) IgG. These studies have provided evidence for the phenomenon of "linked immunological suppression" which may be summarized by the statement "Ts cells specific for an epitope of a given antigen, AgA, suppress the antibody response to an unrelated antigen, AgB, only if the latter is presented in the form of a covalent adduct, AgA-AgB, to the immune system of the animal pretolerized with AgA (mPEG)n, but not if AgB is presented as a mixture with AgA.

Animals↗

PubMed1991

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus.

Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10(5) (approximately 100 x LD50) or 10(6) (approximately 1000 x LD50) tumor cells and received repeated i.p. injections of 25 micrograms anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10(5)/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 micrograms mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.

Animals↗

PubMed1991

Immunologic characterization of a recombinant Kentucky bluegrass (Poa pratensis) allergenic peptide.

A recombinant peptide of Kentucky bluegrass (KBG) pollen was synthesized as a fusion protein (FP) in Escherichia coli by recombinant DNA procedures and was compared with its natural counterparts with respect to its allergenic properties. The FP was demonstrated to bind to the IgE antibodies (Abs) of greater than or equal to 95% of 55 individual sera examined. A positive correlation (r = 0.90) was observed between the levels of IgE Abs corresponding to the FP and the grass-pollen extract(s). With sera of five allergic patients, the IgE binding of three different protein preparations were compared, namely, KBG pollen proteins, 27 to 35 kd gel-purified pollen proteins, and the FP. Results indicated that about 50% of the total IgE binding of KBG pollen proteins was due to the IgE Abs specific to FP. Comparison of the above protein preparations with respect to their abilities to specifically stimulate murine popliteal lymph node cells in vitro indicated that the total pollen proteins stimulated the highest proliferation of lymph node cells. Interestingly, the FP supported higher proliferation of lymph node cells than the gel-purified proteins. Collectively, these results suggest that the recombinant peptide constitutes a major allergenic constituent of grass pollens and may be of diagnostic and therapeutic value.

Allergens↗

PubMed1991

A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

Allergens↗

PubMed1991

Allergenic and antigenic cross-reactivities of group IX grass pollen allergens.

The allergenic and antigenic cross-reactivities between a major recombinant Poa pratensis (Poa p) IX allergen, rKBG8.3, and its corresponding proteins of different grass pollens were examined. Immunoblotting of the proteins of thirteen different grass pollens using anti-rKBG8.3 antibodies indicated that Poa p IX-like proteins are present in ten other grass pollens, albeit in variable amounts and polymorphic forms. These proteins ranged in size from 20 to 88 kDa in different grass pollens. The percent relative binding determined for each grass pollen extract using allergic human sera showed a significant correlation (r = 0.891) with that of anti-rKBG8.3 antiserum. Moreover, there was a strong association (r = 0.901) between the Kentucky bluegrass extract and rKBG8.3 with respect to their inhibition of the binding of human IgE antibodies to allergens in grass pollen extracts. Taken together, these results suggest that the allergenic and antigenic epitopes of the Poa p IX-related proteins in some but not all grass pollens are similar in structure and specificities. It is concluded that the group IX allergens constitute a major family of homologous proteins in several grass pollens.

Allergens↗

PubMed1991

Carl Prausnitz Memorial Lecture. Suppression of antibody responses by chemically modified antigens.

Some of the studies employing nonantigenic, nonallergenic and tolerogenic derivatives of antigens and allergens, synthesized by coupling onto them an optimal number (n) of molecules of monomethoxy-polyethylene glycol (mPEG), are briefly reviewed. Administration of antigen (mPEG)n conjugates into mice resulted in specific immunosuppression, which was mediated by antigen-specific suppressor T (Ts) cells and by suppressor factor(s) (TsF) produced by these cells. These Ts cells were cloned and shown to be Thy-1.2+, CD3+, CD4-, CD5-, CD8+, and expressed the alpha beta heterodimer of conventional T cell receptors (TCR). The TsF had the functional activity of Ts cells and possessed at least one epitope related to the alpha-chain of TCR. The results of clinical trials of mPEG conjugates of common allergens are briefly referred to; it is suggested that the time is opportune for the evaluation of the therapeutic efficacy of mPEG conjugates of pure allergens which can be synthesized on an industrial scale by recombinant DNA technology. Finally, possible applications of tolerogenic mPEG conjugates of xenogeneic monoclonal antibodies, immunotoxins and immunogenic recombinant lymphokines to the development of immunotherapeutic strategies in oncology, transplantation, autoimmunity and acquired immunodeficiency syndrome are discussed.

Allergens↗

PubMed1990

Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol. Relationship between monoclonal T suppressor factor and the T cell receptor.

Cloned Ts cells specific for the Ag, human monoclonal (myeloma) IgG, were derived from spleen cells of mice that had been immunosuppressed by treatment with a tolerogenic conjugate of HIgG and monomethoxypolyethylene glycol. The cloned Ts cells (clone 23.32) suppressed in vitro antibody responses in an Ag-specific and MHC-restricted manner. By FMF with appropriate antibody reagents, these cells were shown to be Thy-1+, CD4-, CD5-, and CD8+ and to express CD3 and the alpha beta-TCR. These results are consistent with the view that Ts cells use Ag recognition structures similar to those reported for Th cells and CTL. A soluble factor (TsF) extracted from the cloned Ts cells also suppressed in vitro antibody responses in an Ag-specific and H-2Kd-restricted manner, i.e., restricted to MHC class I molecules. The suppressive activity of this TsF could be abrogated by addition of mAb H28-710 that reacts with a determinant on the alpha-chain of TCR. Moreover, the TsF bound to and could be recovered from an immunosorbent consisting of the anti-alpha-TCR mAb H28-710 coupled to Sepharose 4B. In contrast, the TsF was not bound by immunosorbents consisting of mAb to the beta-chain of TCR (H57-597) or to V beta 8 (F23.1). It was, therefore, concluded that the TsF of clone 23.32 is serologically related to the alpha-chain of the TCR; however, it is not identical to TCR, because it lacks the determinants expressed on the TCR beta-chain that are recognized by the two anti-beta mAbs used in this study.

Animals↗

PubMed1990

Isolation and characterization of a cDNA clone encoding an IgE-binding protein from Kentucky bluegrass (Poa pratensis) pollen.

We reported previously on the isolation and characterization of several allergens from Kentucky bluegrass (KBG) (Poa pratensis L.) pollen with the aid of the corresponding murine monoclonal antibodies (Mabs). In the present study, (1) an analysis of various tissues of this grass revealed that the allergenic components recognized by these Mabs were confined to the pollen; (2) intact translatable mRNA was isolated from the KBG pollen, and (3) a cDNA library was constructed with this mRNA in the lambda gt11 expression vector. Screening of this library with a pool of six sera from KBG-allergic patients, in combination with enzyme-labeled antibodies to human IgE, led to the isolation of a cDNA clone, referred to as KBG7.2. The nick-translated cDNA probe of KBG7.2 hybridized to a 1.5-kbp RNA transcript from KBG pollen. Moreover, transcripts corresponding to KBG7.2 were found in pollens of eight other grasses, indicating that the proteins similar to the one encoded by this cDNA may be present in these grasses. The nucleotide sequence of KBG7.2 was determined; interestingly, the corresponding derived amino acid sequence did not match any other sequence recorded in the protein data banks. The peptide encoded by KBG7.2 was expressed as a fusion protein utilizing the plasmid vector pWR590.1. Whereas none of the above allergen-specific Mabs bound to the fusion protein, all the 15 individual sera from grass pollen allergic patients recognized the fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

Modulation of antibody responses by conjugates of antigens with monomethoxypolyethylene glycol.

The use of tolerogenic conjugates of monomethoxypolyethylene glycol (mPEG) and diverse antigens, including xenogeneic monoclonal immunoglobulins, is reviewed with respect to the induction of specific abrogation of antibody responses. Evidence is presented for activation of specific suppressor T (Ts) cells by antigen-mPEG conjugates and for the release of suppressor lymphokines (TsFs) from these cells; both Ts cells and TsFs are able to suppress antigen-specific immune responses on transfer to syngeneic mice. The therapeutic potential of tolerogenic mPEG conjugates is discussed with respect to IgE-mediated allergies and diseases which could be ameliorated by administration of xenogeneic monoclonal antibodies and of their respective immunotoxins, as would be the case for suppression of rejection of organ transplants, localization and destruction of tumors, and in vivo annihilation of the human immunodeficiency virus (HIV).

Animals↗

PubMed1989

Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV.

Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.

Allergens↗

PubMed1989

Down-regulation of secondary in vitro antibody responses by suppressor T cells of mice treated with a tolerogenic conjugate of ovalbumin and monomethoxypolyethylene glycol, OVA(mPEG)13.

In previous studies from this laboratory it was shown that OVA(mPEG)n conjugates induced: (i) tolerance in mice with respect to IgG and IgE antibody responses to dinitrophenylated OVA (DNP-OVA); and (ii) OVA-specific suppressor T (Ts) cells which could down-regulate a primary immune response in vivo. For the present study, we have developed an in vitro culture system for assessing the activity of Ts cells of mice tolerized by an OVA(mPEG)13 conjugate. Spleen cells from mice which had been primed with DNP4-OVA in Al(OH)3 gel were cultured with DNP4-OVA to induce a secondary antibody response in vitro. After 6 days, cells secreting anti-DNP antibodies of the IgG1 class were enumerated by an immunoenzymatic plaque-forming cell assay. Addition to the culture of T cells from mice treated with 3 i.p. injections of 500 micrograms of OVA(mPEG)13 resulted in a 29-61% reduction in the number of IgG1 anti-DNP antibody-forming cells, in comparison with the effect of T cells from mice treated with PBS. It was concluded that this tolerogenic conjugate induced splenic Ts cells which were capable of suppressing secondary in vitro anti-DNP responses.

Animals↗