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H Lemke

Publications and source records attributed to H Lemke.

At least 19 recordsLinked to original sources
PubMed2000

CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme.

CD30 is a costimulatory receptor on activated lymphocytes and a number of human lymphoma cells. Specific ligation of membrane-bound CD30 or cellular stimulation by PMA results in a metalloproteinase-mediated down-regulation of CD30 and release of its soluble ectodomain (sCD30). In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was mediated by a membrane-anchored metalloproteinase which was active on intact cells following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate extraction of membrane preparations. Moreover, CD30 shedding was blocked by the synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC(50), 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 (IC(50), 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2. This inhibition profile is similar to that of the TNF-alpha- converting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound metalloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-TACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitantly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-alpha, but also that of sCD30.

ADAM Proteins↗
PubMed2000

Announcements

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Journal Article↗
PubMed2000

Announcements

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Journal Article↗
PubMed1999

A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene.

A new phagemid cloning vector for positive selection of recombinants, pBa-7, was constructed which contains an active barnase gene encoding the cytotoxic ribonuclease from Bacillus amyloliquefaciens, under control of the lac promoter. PBa-7 is a derivative of the high-copy number pBluescript II KS+ phagemid in which the modified barnase killer gene has been fused downstream from the lac promoter of the pBluescript II KS+ multiple restriction site. When a lacIq-negative Escherichia coli strain is transformed by this vector, the active barnase blocks bacterial growth by massive RNA destruction [1]. However, if barnase is inactivated by insertion of a foreign DNA fragment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are considerably simplified. When E. coli transformants are plated out on rich medium with ampicillin, only cells containing recombinant plasmids give rise to colonies. In a lacIq-positive host, the positive selection is IPTG-dependent. Therefore, pBa-7 phagemid can be amplified and prepared in large quantities from lacIq-positive E. coli hosts. Hence, pBa-7 seems to be suitable for most genetic engineering manipulations.

Ampicillin↗
PubMed1999

An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line.

The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). The resulting immunotoxin Ki-4(scFv)-ETA' specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies.

Bacterial Toxins↗
PubMed1999

Genetic analysis of the maternally induced affinity enhancement in the non-Ox1 idiotypic antibody repertoire of the primary immune response to 2-phenyloxazolone.

The early phases of ontogeny are decisive for the development of the B-cell repertoire. Here, we demonstrate that maternal tertiary immunization of BALB/c mice with 2-phenyloxazolone (phOx) caused a drastic alteration of the primary antigen-specific repertoire of the offspring. Maternal tertiary immunization or quaternary antibodies, which exhibited an extremely weak cross-reactivity with the major Ox1 idiotype (IdOx1), induced a change in the proportion of IdOx1/non-IdOx1 antiphOx antibodies in the F1 and F2 primary repertoire. The observed variability in the level of IdOx1 expression (10-90%) exceeded even the seemingly genetically based differences between various mouse strains. In comparison with the non-IdOx1 of control mice, half of the non-IdOx1 antibodies showed a 5-100-fold enhanced affinity. Sixty per cent of these antibodies exhibited an affinity identical to that of IdOx1 antibodies, which are normally of the highest affinity, while the remaining 40% exceeded even that of IdOx1 by a factor of 10. The non-IdOx1 were encoded by VH/VL genes and/or combinations thereof which are either new, hitherto unobserved in the antiphOx response, or typical of memory responses in normal mice. The significance of these data is discussed with respect to the possibility that maternal antibodies, which are acquired through multiple immune maturation processes, may have an epigenetic (non-Mendelian) inheritable potential for the offspring.

Amino Acid Sequence↗
PubMed1999

Is there a maternally induced immunological imprinting phase à la Konrad Lorenz?

In mammals, IgG antibodies are transferred from mothers to the offspring. Since these maternal antibodies result mainly from thymus-dependent immune responses which have undergone immune maturation through somatic hypermutations, they represent the highest quality of the collective maternal immunological experience. Maternal antibodies not only confer passive immunity as long as the newborn's immune system has not fully developed, but also exert an active stimulation as indicated by their regulatory influence on isotype expression, long-term idiotypic alterations, determination of the adult B and T cell repertoire, induction of antigen reactive IgM as well as an affinity enhancement of a proportion of early primary antibodies. The fact that several of these features can only be induced during limited sensitive periods shortly after birth is reminiscent of the behavioural imprinting as defined by Konrad Lorenz. We therefore propose that during early ontogeny there is an immunological imprinting phase with characteristics analogous to behavioural imprinting: (i) the internal imprinting effect is induced by external signals, (ii) in contrast to normal learning, immunological imprinting is also only possible during certain development phases and (iii) it is characterised by an (almost) irreversible result. Hence, if particular immunological experiences are only possible during such sensitive phases, maternal immunoglobulins and consequently the mother's immunological experience is of prime importance for the start of the ontogenetic development of the immune system.

Animals↗
PubMed1998

Antigen-independent suppression of the IgE immune response to bee venom phospholipase A2 by maternally derived monoclonal IgG antibodies.

The IgE immune response to ovalbumin in rats can be suppressed by prior immunization of the dams. The results reported in this paper extend this observation to include a different antigen and another species, namely the IgE immune response to bee venom phospholipase A2 (PLA2) in CBA/J mice. The degree of suppression seemed to depend on the amount of IgG antibodies transferred to the offspring. Moreover, we found that the maternally mediated suppression of the IgE response could be achieved in a completely antigen-free system in which exogenous monoclonal anti-PLA2 IgG antibodies were transferred from the dams to the offspring. The following results were obtained: (i) The IgE suppression by monoclonal IgG antibodies was induced as efficiently with one single anti-PLA2 IgG1 antibody as with a mixture of ten antibodies (nine IgG1, one IgG2b). (ii) Even after several immunizations up to an age of 6 months with a dose of PLA2 that normally induces IgE production, none of the F1 mice developed an IgE response. (iii) This long-lasting suppression was observed in mice which were first immunized at an age of 4 weeks (i.e. when low amounts of maternally derived monoclonal IgG were still present), as well as in mice which were first immunized at an age of 8 weeks, when no such maternal antibodies could be detected in their sera. The corresponding IgG responses showed, compared to normal mice, a transient enhancement in the maternally influenced mice. It is concluded that the immunological experience of the mother is of particular importance for the isotype regulation in the newborns, especially with respect to the ability to elicit an IgE response. The possible implications for the development of allergic diseases in humans are discussed.

Animals↗
PubMed1997

High plasma levels of the soluble form of CD30 activation molecule reflect disease activity in patients with Wegener's granulomatosis.

PURPOSE: To determine the plasma levels of soluble CD30 (sCD30) in Wegener's granulomatosis (WG) patients, and to investigate the possible correlation of sCD30 with disease extent and activity. PATIENTS AND METHODS: sCD30 was determined by radioimmunoassay in 57 WG patients, 25 patients with rheumatoid arthritis (RA), 23 patients with bacterial infections and 21 healthy controls (HC). The extent and activity of WG disease were assayed according to disease extent index (DEI) and standard laboratory parameters. RESULTS: Plasma sCD30 levels in generalized WG (22.5 +/- 1.5 U/mL), but not in initial phase WG (12.1 +/- 4.0 U/mL), were significantly increased compared with HC (8.8 +/- 0.9 U/mL, P < 0.0001). Furthermore, of 11 generalized WG patients who received long-term follow-up, sCD30 levels declined when the disease activity changed from active disease to remission (29.1 +/- 1.9 U/mL to 15.9 +/- 1.8 U/mL, P = 0.0001). Similar results were observed in the whole group of generalized WG, eg, sCD30 levels in active disease (29.4 +/- 1.4 U/mL) were significantly higher than in partial remission (17.9 +/- 1.9 U/mL, P < 0.001) and in complete remission (13.7 +/- 3.3 U/mL, P < 0.001). No significant difference was noted between complete remission and HC. In addition, sCD30 levels were correlated with other parameters of disease extent and activity such as DEI, plasma levels of sIL-2R, PR3-ANCA, ESR and CRP. The sCD30 levels were increased in RA patients compared with HC (15.2 +/- 2.1 U/mL, P < 0.05), but no correlation was found between disease activity parameters and sCD30 levels. In contrast, in patients with bacterial infections sCD30 levels (6.9 +/- 0.9 U/mL) were not significantly different compared with HC. CONCLUSION: Plasma levels of sCD30 are not only significantly increased but also correlate with disease extent and activity in generalized WG. These findings suggest that sCD30 can act as a useful marker for evaluation of disease extent and activity, and that generalized WG may be associated with Th2-type immune response.

Arthritis, Rheumatoid↗
PubMed1997

Characterization, mapping and partial cDNA sequence of the 57-kD intracellular Ki-1 antigen.

A novel antigen was identified by the cross-reactivity of the anti-CD30 antibody Ki-1. This 57-kD intracellular Ki-1 antigen (Ki-1/57) is induced upon activation of leukocytes and is transported to the nuclear compartment. We describe the partial cloning and sequencing of the Ki-1/57 cDNA from a lambda gt 11-cDNA library derived from the Hodgkin-analogous cell line L540. New monoclonal antibodies were produced against the recombinant Ki-1/57 protein fragment which were used to confirm that the Ki-1/57 antigen is associated with kinase activity and is expressed in a variety of tumor cell lines and in activated but not resting leukocytes. The Ki-1/57 gene was mapped to the bands 9q22.3-31 of human chromosome 9. This is an area which appears to be associated with secondary chromosomal aberrations in acute myeloid leukemias.

Amino Acid Sequence↗
PubMed1996

Correlation between immune maturation and idiotypic network recognition.

The maturation of T-dependent humoral immune responses is mediated by somatic mutations. Antigen selection is one mechanism for the activation of B cell clones which express antibodies with progressively increased affinity and which are derived as somatic variants from germ-line-encoded genes. However, the emergence of B cell clones secreting rather low-affinity antibodies and the shift to alternative germ-line V region gene combinations during secondary and tertiary responses cannot be explained by antigen selection. It has been considered that idiotypic suppression may favor this clonal shift. Such an involvement would require that idiotypic recognition in the syngeneic host must be highly restricted to private idiotopes of each clone sequentially activated during immune maturation. To test this possibility, we produced 19 syngeneic anti-idiotypic antibodies to the germ-line-encoded major Ox1 idiotype (IgM-IdOx1 H11.5) of the anti-2-phenyl-oxazolone (phOx) immune response in BALB/c mice. The fine specificity of these anti-IdOx1 was tested with a set of anti-phOx monoclonal antibodies, representing the first steps of maturation. About half of the anti-IdOx1 showed almost no reactivity with the IdOx1 after the switch to IgG and none of the anti-IdOx1 reacted with anti-phOx antibodies which carried a glycine or histidine instead of arginine as the middle amino acid of the D region. These observations suggest a strong correlation between immune maturation and the idiotypic network. A model is presented in which idiotypic suppression may function as a driving force for diversification and maturation of the antigen-induced immune response.

Animals↗
PubMed1996

Induction of a non-oscillating, long-lasting humoral immune response to an internal network antigen.

B lymphocyte antigen receptors form an internal idiotypic network which is also connected by idiotypic interactions with the T lymphocyte compartment. Idiotypic-anti-idiotypic activation of lymphocytes has mainly been measured at the cellular level while the kinetics of primary anti-idiotypic humoral responses has so far not been determined. Here, we describe the induction of an anti-idiotypic immune response to the major idiotype (IdOx1) of the primary immune response in BALB/c mice to the hapten 2-phenyl-t-oxazolone coupled to chicken serum albumin. A primary anti-idiotypic humoral response could be induced with the phOx-binding and IdOx1-expressing, germline-encoded antibody H11.5 (mu, kappa) coupled to keyhole limpet hemocyanin. Compared to that of conventional antigens, the anti-idiotypic response showed a lag phase of 3 weeks. When the anti-IdOx1 serum titers had declined to background levels, a secondary anti-IdOx1 response could be induced even with soluble H11.5. This response showed as fast an increase as conventional antigens, but the antibody plateau did not exceed that of the primary response. During this secondary anti-IdOx1 response and probably to a small extent also during the primary response, the mice developed an idiotypically non-related IgM-anti-phOx response. In contrast, soluble H11.5 - either passively injected or transiently expressed during the early primary anti-hapten response - suppressed the anti-idiotypic response to H11.5 for up to 7 months in the majority of mice, while individual mice exhibited an early release from this suppression at various times. The differences and similarities between external and internal antigen-induced immune responses as well as the implications for idiotypic network regulation are discussed.

Animals↗
PubMed1995

A zinc metalloproteinase is responsible for the release of CD30 on human tumor cell lines.

The activation marker CD30 is expressed on the cell surface of the malignant cells in Hodgkin's disease and a few non-Hodgkin lymphomas. We have analyzed the regulation of membrane-bound CD30 and found that the binding of a variety of anti-CD30 antibodies induced down-regulation of CD30 on cell lines. In addition, such down-modulation was also observed after treatment of the cell surface proteins with the sulfhydryl reagent iodoacetamide or after stimulation of the second messenger pathway with phorbol ester or calcium ionophore. This modulation was abolished at 4 degrees C and strongly inhibited by chelators like EDTA or 1,10-phenanthroline, whereas EGTA, a selective inhibitor of Ca(2+)-dependent proteinases and other inhibitors of serine, thiol and acid proteinases, showed no effect. The down-modulation was strengthened by Zn2+ or Cd2+, but not by other divalent cations such as Fe2+, Mn2+, Mg2+, Ca2+ or Co2+, thus indicating the involvement of a zinc metalloproteinase in CD30 modulation which can be activated by protein kinase C and by alkylation of sulfhydryl groups. Pulse-chase experiments, analysis of the CD30 glycosylation and specific measurement of the 90-kDa soluble form of CD30 (sCD30) with a sandwich radioimmunoassay revealed that CD30 down-modulation results from enhanced release of 90-kDa sCD30 by the site-specific cleavage of CD30 accomplished by a zinc metalloproteinase. This release occurs at the cell membrane without prior endocytosis.

Antibodies, Monoclonal↗
PubMed1995

Development of new ricin A-chain immunotoxins with potent anti-tumor effects against human Hodgkin cells in vitro and disseminated Hodgkin tumors in SCID mice using high-affinity monoclonal antibodies directed against the CD30 antigen.

The lymphocyte activation marker CD30 has been shown to be an excellent target for the immunotherapy of human Hodgkin's lymphoma. In order to develop new potent immunotoxins (ITs) against CD30, we chemically linked 6 recently described monoclonal antibodies (MAbs) via SMPT to deglycosylated ricin A-chain (dgA). Cross-blocking experiments demonstrated that these MAbs, termed Ki-2 to Ki-7, recognize 3 different clusters on the CD30 antigen: Ki-2, Ki-4, Ki-5 and Ki-7 recognize cluster A; Ki-6 recognizes cluster B; Ki-3 binds to cluster C. Staining of 29 sections of normal human organs revealed no major cross-reactivity of any MAbs tested. Binding to the CD30 antigen on L540Cy Hodgkin cells was assessed by flow cytometry, and demonstrated high affinities for Ki-2, Ki-3 and Ki-4. The concentration giving 50% of the mean fluorescence intensity (MFI50) was 0.58 micrograms/ml to 0.78 micrograms/l. MAbs Ki-5, Ki-6, and Ki-7 bound much more weakly. The staining intensity of the MAbs correlated with the cytotoxicity of the corresponding ITs. Ki-2.dgA, ki-3.dgA and Ki-4.dgA inhibited the protein synthesis of L540Cy cells by 50% at concentrations (IC50) of 3.5 x 10(-10)M to 4.0 x 10(-11)M. The most effective IT, Ki-4dgA, is 5-fold more potent than previously reported CD30 ricin A-chain ITs. Ki-4.dgA was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID mouse model. The mean survival time (MST) of lymphoma-bearing SCID mice was extended from 42 days in untreated controls to more than 132 days when Ki-4.dgA was applied one day after tumor challenge. Ki-4.dgA is a new potent IT suitable for further evaluation against Hodgkin's lymphoma in man.

Animals↗
PubMed1995

Shedding of the soluble form of CD30 from the Hodgkin-analogous cell line L540 is strongly inhibited by a new CD30-specific antibody (Ki-4).

The CD30-activation marker was detected as the Hodgkin-associated Ki-I antigen and is regarded as a target for the treatment of Hodgkin patients with immunotoxins. The CD30 is released from tumor cells and this soluble CD30 (sCD30) is an indicator of the disease activity. Since the shedding of sCD30 may be influenced by antibodies, we produced 6 new CD30-specific antibodies (Ki-2 to Ki-7) for the purpose of finding antibodies that might inhibit the formation of sCD30. Ki-2 to Ki-7 and the other anti-CD30 antibodies Ki-I, Ber-H2, HeFi-I, M44, M67, HRS-I, HRS-4 and C10 were employed for epitope mapping. The binding of a particular radio-labeled anti-CD30 antibody to Hodgkin's-disease-derived L540 cells was completed by addition of the various non-labeled anti-CD30 antibodies. Three non-overlapping regions, expressing different antigen-specific determinants, could be defined on the extracellular part of the CD30 molecule. Cluster A of determinants was recognized by Ki-2, Ki-4, Ki-6 and Ki-7, Ber-H2, HRS-I and HRS-4, while cluster B was detected by Ki-I, Ki-5 and M67. Cluster C, which probably contains the binding site for the CD30 ligand, was defined by Ki-3, M44, HeFi-I and C10. Co-culture experiments of L540 cells with the various antibodies followed by the isolation of sCD30 from culture supernatant fluids revealed that the release of sCD30 was most strongly increased by Ki-I and weakly enhanced by Ki-2, Ki-3, Ki-5 and HeFi-I, whereas it was almost completely inhibited by Ki-4 and to a slightly lesser extent by Ber-H2.

Animals↗
PubMed1995

Molecular cloning and characterization of human kinectin.

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.

Amino Acid Sequence↗
PubMed1994

Maternal immunization modulates the primary immune response to 2-phenyl-oxazolone in BALB/c mice.

The development of the antibody repertoire in newborn mice is greatly influenced by idiotype network interactions. It has been demonstrated that anti-idiotypic antibodies either directly injected or transferred from the mother may alter the repertoire for life. For an elucidation of the underlying mechanisms we have analyzed the primary immune response to 2-phenyl-5-oxazolone (phOx) coupled to chicken serum albumin (CSA) in BALB/c mice after complete disappearance of maternal antibodies which originated from different stages of affinity maturation. Depending on the serum titers of the mothers after primary (1 degree mo), secondary (2 degrees mo) or tertiary (3 degrees mo) immunization, maternal anti-phOx IgG persisted in F1 mice for up to 9 months. In addition, F1 mice born to 2 degrees mo developed--even without immunization--an anti-phOx IgM titer which reached levels similar to an antigen-induced primary response. An enhancement of the early primary anti-phOx as well as anti-CSA response was seen in F1 mice born from 1 degree mo, whereas the response was delayed when born to 2 degrees mo and 3 degrees mo. The antibody titers in the latter group of mice remained at a lower level for 3 months. In contrast, mice of the F2 generation which received a smaller amount of the same collection of maternal antibodies as F1 mice from 3 degrees mo exhibited a quite different primary response: (i) They showed an earlier onset in their anti-CSA response. (ii) Whereas normally a plateau in antibody titer was reached by the 4th weak after immunization, in 55% of the F2 mice a prolonged increase of the anti-phOx and anti-CSA antibody titers was observed. At 12 weeks after antigenic challenge, titers reached plateau levels of 6 x 10(5) which were never before seen in a primary phOx or CSA response. Thus, depending on its own immunological experience, the maternal immune system induces a state of memory in the offspring which results in a faster and/or enhanced immune response in the F1 and F2 [corrected] generations.

Animals↗