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Biomedical subjects

H Kohler

Publications and source records attributed to H Kohler.

At least 73 records · Page 4Linked to original sources
PubMed1988

Biological mimicry of antigenic stimulation: analysis of the in vivo antibody responses induced by monoclonal anti-idiotypic antibodies.

In this study, the induction of protective antibodies against a bacterial pathogen in mice was used as a model for idiotype vaccine development. The antibody responses induced in different strains of mice by the hapten phosphorylcholine (PC) coupled to ovalbumin, PC-OVA, were compared with the responses induced by carrier conjugates of two different anti-idiotopic antibodies. One anti-idiotope, 4C11, exhibits the characteristics of an internal image of phosphorylcholine, and therefore is classified as an Ab2 beta; the other, F6, does not mimic antigen, and therefore is classified as an Ab2 alpha. The analysis of the temporal kinetics of the IgM and IgG1 anti-PC responses induced by nominal and idiotope antigens revealed dynamic responses characterized by changes in the quality and quantity of the antibody populations during the course of the immune response. All three antigens could stimulate antibodies that were PC-specific and T15 idiotope-positive in BALB/c and A/St mice. The highest titre of T15+ anti-PC antibodies was achieved with an immunization protocol which involved priming with Ab2 alpha followed by challenge with PC-OVA. Antibodies specific for the extended hapten, diazophenylphosphorylcholine, and hapten-carrier bridge determinants were being stimulated late in the responses to PC-OVA. BALB-c, A/St and CBA/N (Xid) mice all produced, late in the response to Ab2 alpha, high T15+ antibody titres which do not bind PC. The induction of T15+, non-PC binding, antibody suggests that T15 is a regulatory idiotope, expressed on antibodies having differing antigenic specificities. With regard to vaccine development, these results support the contention that effective induction of antibodies does not depend on stimulating a unique idiotope but can be achieved by anti-idiotypes reacting with different idiotopes. In addition, these results suggest that the combined use of idiotope and nominal antigens in an immunization protocol may provide the maximal protective immunity.

Animals↗
PubMed1987

Analysis of the idiotypic network in tumor immunity.

Herein we have analyzed the expression of idiotopes associated with a monoclonal anti-tumor-associated antigen (TAA) antibody in DBA/2 mice which have progressively growing tumors or resist tumor growth. A panel of eight monoclonal antiidiotypic antibodies raised against a monoclonal antibody which reacts with a mouse mammary tumor virus cross-reactive qp52 envelope protein (TAA) of the L1210/GZL lymphoma was used to measure the expression of idiotopes in sera from different treatment groups. Significant correlations between the expression of certain idiotopes and the growth of the tumor or the establishment of anti-tumor immunity are seen. 1) Idiotypes detected by anti-idiotype D11 are high in anti-idiotype immunized progressor or tumor-susceptible mice and low or absent in regressor mice, i.e., the mice immunized with the protective 2F10 anti-idiotype; 2) the 3A4-detected idiotypes are less frequent or absent in irradiated tumor-immunized regressor mice than in untreated mice challenged with live tumor or progressor mice; 3) no difference in the anti-TAA titers is seen in mice in which the tumor growth is inhibited and in mice in which the tumor grows; 4) no difference in 11C1 idiotype + anti-TAA titer was observed between regressor and progressor mice; and 5) mice with normal or accelerated tumor growth have higher titers of idiotypes detected by a polyclonal anti-idiotype. These findings provide evidence for a regulatory idiotype network induced by the growing L1210/GZL tumor or by anti-idiotypic immunization. The titer of anti-TAA antibody does not correlate with the biology of tumor growth, but certain idiotopes correlate with either progressive or regressive tumor behavior. Therefore, the target of the idiotype regulation is likely to be anti-tumor T effector cells. Effective idiotype therapy of tumors must deal with the complexity of idiotype regulation induced by the tumor itself and is unlikely to be successful if anti-idiotypes are used only as internal mimicry of a TAA.

Animals↗
PubMed1987

The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H2O2. A spin-trapping/ESR and absorption spectroscopy study.

H2O2 is catalytically metabolized by ferric lactoperoxidase (LPO)----compound (cpd) I----cpd II----ferric LPO cycles. An excess of the substrate, however, is degraded by a ferric LPO----cpd I----cpd II----cpd III----ferrous LPO----ferric LPO cycle. This latter pathway leads to the partial or total irreversible inactivation of the enzyme depending on the excess of H2O2 (H. Jenzer, W. Jones, and H. Kohler (1986) J. Biol. Chem. 261, 15550-15556). Spin-trapping/ESR data indicate that in the course of the reaction superoxide (HO2./O2-) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-1-pyrroline-N-oxide) desferrioxamine, albumin, or mannitol, do not prevent enzyme inactivation, we conclude that OH. generation is a site-specific reaction at or near the active center of LPO where bulky scavenger molecules may not be able to penetrate. We suggest the formation of OH. by a Fenton-like reaction between H2O2 and the intermediate ferrous state of the enzyme, which substitutes for Fe2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H2O2 to produce HO2. and H2O, or the prosthetic group to give rise to oxidative cleavage of the porphyrin ring structure of the heme moiety of LPO and thus to the liberation of iron.

Electron Spin Resonance Spectroscopy↗
PubMed1987

Idiotope vaccine against Streptococcus pneumoniae. A precursor study.

An analysis of nominal vs idiotope antigen-induced B cell precursors was performed in A/St mice. With the use of the splenic fragment culture system, the quantity and quality of B cell precursors responding to two anti-idiotope carrier antigens (4C11 hemocyanin and F6 hemocyanin) and nominal antigen (phosphorylcholine-hemocyanin) were compared. In addition, the effect of priming with anti-idiotope-carrier antigens on B cell precursors responding to phosphorylcholine-hemocyanin was determined. We found that one anti-idiotope-carrier antigen, 4C11-hemocyanin, and phosphorylcholine-hemocyanin stimulated similar subpopulations of primary B cells. However, the B cell population stimulated by F6-hemocyanin, the other anti-idiotype complex, was distinct. Furthermore, priming with certain idiotope antigens can direct the phosphorylcholine-hemocyanin response into the expression of idiotypes that may be the most effective in protective immunity. Our results provide essential information for the rational design of idiotype vaccines by clarifying the dynamic relationship of the B cell precursor repertoire with the in vivo antibody response in the response to nominal and idiotope antigens.

Animals↗
PubMed1987

Tumor-specific idiotype vaccines. III. Induction of T helper cells by anti-idiotype and tumor cells.

In a previous report, we have demonstrated the induction of tumor-specific immunity by monoclonal anti-idiotype antibodies generated against a monoclonal anti-tumor antibody, 11C1, that also cross-reacts with mouse mammary tumor virus envelope glycoprotein gp52. Also, we showed that whereas one anti-idiotype antibody, 2F10, could induce protective immunity, another anti-idiotype antibody, 3A4, induced nonprotective immunity. Here we demonstrated the existence of T helper cells which recognize anti-idiotypes that exert differential controls on tumor growth. The qualitative nature of idiotype recognizing T cells generated in response to 2F10, 3A4, irradiated tumor, and progressively growing tumor was compared. The reactivity pattern of idiotype recognizing T cells obtained from 2F10 and irradiated tumor immunized mice were similar in nature in the sense that Lyt-2- T cells obtained from these immunized mice responded to both 2F10 and 3A4 as antigen, although T cells from tumor immunized mice responded better to 3A4 antigen. On the other hand, the idiotype-recognizing T cells obtained from 3A4-immunized mice showed a similar reactivity pattern to T cells isolated from mice during the early phase of tumor growth (within day 4 to 5 after the inoculation of 10(4) live tumor cells). Lyt-2- T cells isolated from mice immunized with 3A4 or during the early phase of tumor growth responded only to 3A4 antigen. The inability of Lyt-2- T cells, isolated from 4- to 5-day-old tumor in mice, to cooperate with 2F10-TNP is not due to the absence of 2F10 idiotype recognizing T cells as 2F10 id recognizing T cells are present when examined at the precursor level. These data on the idiotype specificity of T helper cells show a correlation with the presence of anti-tumor immunity. This information will help in the design and application of idiotype vaccine in tumor immunotherapy.

Animals↗
PubMed1987

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes.

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.

Antibodies, Monoclonal↗
PubMed1987

Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta).

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.

Animals↗
PubMed1987

Idiotypic self binding of a dominant germline idiotype (T15). Autobody activity is affected by antibody valency.

We have previously described (1-3) an IgM antibody that binds to PC, expresses the T15 idiotype, and binds also to itself or T15 if insolubilized. Because of the simultaneous presence of complementary idiotopes and paratopes this type of antibody has been termed autobody. The self binding involves the antigen-binding site because the F(ab')2 fragment of T15, PC, and no other haptens inhibit the self binding. DNA sequence analysis of 11E7-1 using primer extension cDNA sequencing showed that the variable sequences of H and L chains of 11E7-1 are identical to the germline sequence of the prototype T15 idiotype. Furthermore, monomeric and dimeric T15 IgA were shown to bind to insolubilized T15 and other T15+ antibodies including 11E7-1. Thus, the self-binding activity is an inherent property of the T15 germline sequence. The self binding is highly dependent on the polymeric state of the binding antibody since the IgM pentamer of 11E7-1 is about three fold more effective than the T15 dimer and 50 times more than the T15 monomer. These data suggest that the self-binding activity of a germline-encoded idiotype may play an important role in the biology of its expression, and more specifically, may be responsible for the establishment of its dominant expression.

Amino Acid Sequence↗
PubMed1987

Irreversible inactivation of lactoperoxidase in the course of iodide oxidation.

In the course of lactoperoxidase-catalysed I- oxidation, which is a model for the initial step of thyroid hormone biosynthesis, irreversible enzyme inactivation can occur if free molecular iodine (I2) or other oxidized iodine species accumulate. Evidence is presented that the breakdown of the catalytic activity is the result of the iodination of the peroxidase-apoprotein. This kind of enzyme inactivation, which can be prevented by iodine acceptors' such as thyroglobulin or high concentrations of I-, may well play a role in the regulation of the synthesis of thyroid hormones in vivo.

Iodides↗
PubMed1987

A disproportionate accumulation of fibrous tissue is not a causal factor in human goitre growth.

Histological preparations from human nodular goitres reveal the presence of variable and sometimes considerable amounts of acellular material separating the individual follicles. Part of this interstitial tissue consists histologically of fibrous strands. However, quantitative data on the fibrous tissue content of goitres are scarce. In the present study the proportion of fibrous tissue in normal human thyroids and human goitres was determined biochemically by measuring their content of collagen, the predominant component of fibrous tissue. Total collagen content increased in parallel to thyroid weight. The relative collagen content, however, decreased slightly but significantly with increasing thyroid weight. The collagen/DNA ratio (= fibrous tissue/cell number ratio) was not higher in goitres than in normal human thyroids. These results indicate that in goitre growth, there is no disproportionate accumulation of fibrous tissue accompanying the multiplication of thyroid follicular cells. They are in line with the earlier findings that despite histological heterogeneity, the main component of nodular goitres is newly generated follicles.

Age Factors↗
PubMed1986

An efficient and general method for sequencing immunoglobulin mRNAs.

By using two oligonucleotide primers, one complementary to a short DNA segment in the first constant region exon and second complementary to a short segment in the variable region, we have developed a highly efficient method for sequencing immunoglobulin mRNAs. Using the first primer full length immunoglobulin-specific cDNA was synthesized and separated on a denaturing acrylamide/urea gel. The desired band was isolated from the gel and sequenced by Maxam/Gilbert method. Using the sequence generated, a second oligonucleotide primer complementary to a short segment in the variable region was synthesized and similarly used to complete the remaining sequence of the variable region, the signal peptide and the 5'-untranslated region. We have used this method to determine the complete nucleotide sequence of heavy and light chain mRNAs from two anti-idiotypic hybridomas. The method is generally applicable to sequencing immunoglobulin mRNAs from both myelomas and hybridomas especially in instances involving the expression of more than one heavy or light chain mRNAs as well as instances involving additional transcripts from aberrantly rearranged immunoglobulin genes.

Amino Acid Sequence↗
PubMed1986

On the molecular mechanism of lactoperoxidase-catalyzed H2O2 metabolism and irreversible enzyme inactivation.

Lactoperoxidase-catalyzed H2O2 metabolism proceeds through one of three different pathways, depending on the nature and the concentration of the second substrate as an e- donor and/or on pH conditions. In the lactoperoxidase (LPO)-H2O2 system, at low H2O2 concentrations and/or alkaline conditions the peroxidatic cycle involves ferric LPO----compound I----compound II----ferric LPO conversion, whereas high H2O2 concentrations and/or acidic conditions favor the ferric LPO----compound I----compound II----compound III----ferrous LPO----ferric LPO pathway. The compound III/ferroperoxidase states are associated with irreversible enzyme inactivation by cleavage of the heme moiety and liberation of iron. It is likely that either singlet oxygen or superoxide and hydroxyl radicals are involved in the attack on heme iron, because inactivation correlates with oxygen production and can be decreased to a certain degree by scavengers such as ethanol, 1-propanol, 2-propanol, or mannitol. In the LPO-H2O2-I- system, the enzyme may also be inactivated by I2 generated in the course of enzymatic I- oxidation (i.e. during ferric LPO----compound I----ferric LPO cycles).

Ferric Compounds↗
PubMed1986

Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen.

The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors.

Animals↗
PubMed1986

The role of superoxide radicals in lactoperoxidase-catalysed H2O2-metabolism and in irreversible enzyme inactivation.

Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme.

Free Radicals↗
PubMed1986

The role of compound III in reversible and irreversible inactivation of lactoperoxidase.

In the presence of iodide (I-, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1-10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I- cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2 per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1 delta gO2), superoxide radicals (O-.2), or consequently formed hydroxyl radicals (OH.).

Free Radicals↗
PubMed1986

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production.

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.

Animals↗
PubMed1986

Immunoglobulin with complementary paratope and idiotope.

A hybridoma antibody (11E7-1) was isolated from a myeloma fusion with nu/nu BALB/c immunized against the T15 idiotype. This IgM antibody exhibited a dual specificity, binding both to PC and to anti-PC antibodies from two idiotype families. Binding to PC and anti-PC antibodies are completely inhibited by PC analogs. Furthermore, the hybridoma antibody binds to itself. Self-binding is also inhibited by PC analogs. From these data, we suggest that 11E7-1 hybridoma antibody has a PC-specific paratope site, and at same time expresses the internal PC antigen idiotope. The term autobody is proposed to signify its self-binding and potential role in autoimmunity. Autobodies may have a unique role in the network of immune system. Furthermore, it may be a model for designing idiotype vaccines.

Animals↗