Biomedical subjects

M Zöller

Publications and source records attributed to M Zöller.

At least 91 records · Page 5Linked to original sources

PubMed1994

[The value of myocardia protection in chronic hypoxic immature rat hearts].

This study examines the efficacy of three methods for myocardial protection during 8 hours of global ischema at 10 degrees C in immature (28 days) rat hearts subjected to lifelong hypoxia afforded by exposure to simulated high altitude. Hearts in group 1 were protected by rapid topical cooling alone, in group 2 by slow pre-arrest cooling with Krebs-Henseleit solution plus topical cooling and in group 3 by coronary perfusion with St. Thomas' Hospital cardioplegia No 2 (STS 2) plus topical cooling. Hearts in groups 4-6 served as controls without hypoxia and were protected accordingly. Parameters of myocardial function (left ventricular pressure, LVP), the metabolic status (myocardial concentration of ATP and creatine phosphate) and endothelial function (response to the vasodilator acetylcholine) were measured. Myocardial protection by rapid topical cooling alone resulted in equal--or significantly improved--postischemic recovery of LVP and endothelial function compared with slow pre-arrest cooling or additional protection with STS 2. The data advocate topical cooling for myocardial protection during surgical correction of cyanotic congenital cardiac disease in early infancy. In this age group, coronary perfusion with cold crystalloid solutions appears to aggravate ischemic endothelial injury.

Animals↗

PubMed1994

Analysis of molecular functions of the tumor metastasis promoting surface molecule CD44v4-v7 using transgenic mice.

In certain circumstances, metastatic tumor cells may mimic the molecular properties and behaviour of lymphocytes. Support for this hypothesis has come from the observation that activated lymphocytes and some tumor cells need a CD44 variant isoform (CD44v) to survive and/or expand in the lymphatic system. CD44 variant (CD44v) isoforms are created by differential splicing from a pool of at least ten variant exons (v1-v10), the encoded sequences of which are absent in the CD44 standard isoform (CD44s). To dissect the molecular interactions of CD44v, transgenic animals have been generated that constitutively express a variant of CD44 containing sequences encoded by exons v4 to v7 on the surface of T cells. Lymphocytes derived from these transgenic animals show accelerated entry into S phase upon antigenic stimulation, and a subpopulation of the cells constitutively express early lymphocyte activation markers. Our data support the hypothesis that the presence of CD44v4-v7 on the surface of T cells mediates intercellular or intracellular processes which result in the promotion of T cells towards a preactivated state.

Animals↗

PubMed1993

The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments.

A method using PCR amplification and primer extension with fluorescent oligonucleotides was developed to analyze T-cell repertoires. The sizes of the hypervariable CDR3-like regions of the murine T-cell antigen receptor beta chains were measured for all possible V beta-J beta combinations. This analysis shows that beta chains are distributed into at least 2000 groups, a value that provides a lower limit to their complexity. The CDR3 sizes appear to be dependent on the J beta and especially the V beta segment used and correlates with amino acid sequence motifs in the corresponding CDR1 region. This feature of T-cell receptors is discussed.

Amino Acid Sequence↗

PubMed1993

A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells.

The activated oncogene c-Ha-ras induces expression of the surface glycoprotein CD44 in cloned rat embryonic fibroblasts (CREF). Induction is transcriptional as shown by transient cotransfections of c-Ha-ras expression constructs and CD44 promoter reporter gene constructs and depends on the presence of an AP-1 binding site at position -110. Increased transcript levels for the standard isoform of CD44 (CD44s) are accompanied by the appearance of alternatively spliced RNAs and the synthesis of variants of CD44 (CD44v). These CD44v molecules differ from the standard type by the addition of sequences in the extracellular portion of the molecules. The occurrence of CD44v molecules in CREF cells upon induction of the CD44 promoter is probably due to leakiness of the splice control in these cells since stable transfection with c-Ha-ras does not alter the CD44v/total CD44 ratio. Upon ras overexpression, however, using an inducible mouse mammary tumor virus-ras construct, a transient increase of CD44v/total CD44 ratio of 3-4 has been determined suggesting that a burst of ras expression, in the genetic background of CREF cells, influences both promoter activity and splice control or accuracy. The expression of CD44v proteins is responsible for the metastatic potential in a variety of tumors (U. Günthert et al., Cell, 65: 13-24, 1991). Also in CREF cells expression of CD44v correlates with metastatic behavior, ras-transfected CREF cells are not only fully transformed but also give rise to metastatic spread as measured in the spontaneous metastasis assay. The adenoviral oncogene E1A counteracts ras-induced promoter function and, consequently, inhibits metastatic behavior without extinguishing transformation.

Animals↗

PubMed1993

The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic behavior.

The metastatic pancreas carcinoma cell line BSp73ASML produces a variety of different splice variants of the transmembrane glycoprotein CD44. The NH2-terminal portions are identical and heavily glycosylated. The variant sequences are inserted just outside the transmembrane region of the molecules. The two most abundant variants have 162 and 85 extra amino acids, respectively. When individually expressed, these suffice to establish metastatic properties in the nonmetastatic tumor cell line BSp73AS, as assayed by the spontaneous metastasis protocol.

Adenocarcinoma↗

PubMed1993

Prevention of tumor metastasis formation by anti-variant CD44.

A splice variant of CD44 (CD44v) originally discovered on metastases of a rat pancreatic adenocarcinoma (BSp73ASML) has been shown by transfection to confer metastatic behavior to nonmetastatic tumor cells (Günthert U., M. Hofmann, W. Rudy, S. Reber, M. Zöller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. Cell. 65:13). A monoclonal antibody (mAb), 1.1ASML, to the metastasis-specific domain of the CD44v molecule retards growth of lymph node and lung metastases of the metastatic tumor line BSp73ASML, and can efficiently prevent formation of metastases by the transfected line. The antibody is only effective when given before lymph node colonization. Anti-CD44v does not downregulate the expression of CD44v, and prevention of metastatic growth by anti-CD44v is not due to activation of any kind of immune defense. We suggest that the mAb interferes with proliferation of metastasizing tumor cells in the draining lymph node, most probably by blocking a ligand interaction. The interference with metastatic spread will greatly facilitate the exploration of the function of CD44v and, in particular, may also open new strategies for the therapy of human metastases.

Animals↗

PubMed1993

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy.

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and 67Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%-40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.

Animals↗

PubMed1993

CD44 splice variants: metastases meet lymphocytes.

Recent discoveries of surface proteins involved in tumor metastasis formation have revived an old hypothesis that tumor cells may acquire, and use for their metastatic spreading, properties which lymphoid cells had developed to defend the organism against foreign antigens. Splice variants of CD44 and integrins are expressed on metastasizing tumor cells and also on leukocytes at defined stages of their differentiation. Expression and function appear to be essential not only for the generation of an immune response but also for the establishment of metastatic tumor colonies.

Alternative Splicing↗

PubMed1993

Expression of CD44 isoforms carrying metastasis-associated sequences in newborn and adult rats.

Expression of a splice variant of CD44, recognised by the monoclonal antibody (Mab) 1.1ASML, confers metastatic potential to non-metastasising tumour cells (Cell 1991, 65, 13-24). To explore whether the metastasis-associated variant of CD44 (CD44v) is expressed under physiological conditions, tissues of newborn and adult rats were stained with the Mab 1.1ASML. The 1.1ASML epitope is, indeed, expressed on the basal layer of the epidermis and the hair follicles as well as on cryptic epithelia in the gut. In addition, ductal epithelia of the pancreatic gland of newborn rats express CD44v. This pattern of expression differs from that of standard lymphocyte CD44 (CD44s). The anti-CD44s mAB Ox50 predominantly stains connective tissue. Although different variants of CD44 may express the epitope recognised by 1.1ASML, cells expressing CD44v share properties with metastasising tumour cells: the stage of proliferation and a restricted degree of mobility. Thus, during metastatic progression tumour cells may reactivate the expression of gene segments which serve highly specialised functions in embryonic and adult tissues.

Aging↗

PubMed1992

Participation in normal immune responses of a metastasis-inducing splice variant of CD44.

A variant of the glycoprotein CD44 (CD44v) that shares sequences with variants causally involved in metastasis formation is transiently expressed on B and T lymphocytes and macrophages after antigenic stimulation and in the postnatal period. Antibodies to the variant hinder in vivo activation of both B and T cells. The observation that a protein domain that is expressed on CD44 and required for the lymphatic spread of tumor cells can catalyze an essential step in the process of lymphocyte activation supports the idea that metastasizing tumor cells mimic lymphocyte behavior.

Animals↗

PubMed1992

Reduced tumorigenicity of fibrosarcomas which constitutively generate IL-1 alpha either spontaneously or following IL-1 alpha gene transfer.

Interleukin-1 (IL-1) is a major immunoregulatory/proinflammatory cytokine which also affects fibroblast proliferation and function and therefore it was of interest to investigate whether its constitutive expression influences the in vivo tumorigenic potential of transformed fibroblastoid cell lines. Here we report on a strong correlation between the constitutive expression of IL-1 alpha and reduced tumorigenicity, using various series of oncogene-transformed NIH/3T3-derived cell lines which produce the cytokine spontaneously or upon gene transfer, following transfection with the IL-1 alpha cDNA. Reduced tumorigenicity of the constitutive IL-1 alpha producing cell lines was manifested either by inability to grow in animals or by regressions of initially growing tumors, within 2 to 3 weeks from cell inoculation. In contrast, mice inoculated with non-IL-1-producing cell lines developed progressive tumors which ultimately killed the animals. Clones obtained from a non-IL-1-producing met-transformed cell line shifted from a progressive to a regressive phenotype, following transfection with an IL-1 alpha-encoding gene, inserted into an appropriate expression vector, resulting in constitutive expression of the cytokine. The effects of constitutive IL-1 expression on tumor development were observed both in histocompatible (NFS/N) and partially allogeneic (BALB/c) mice; however, they were more pronounced in the allogeneic environment. Fibrosarcomas which are non-IL-1 producers induced progressive tumors in both strains of mice at the same growth rate. The differences between the growth characteristics of the fibrosarcomas in histocompatible vs. partially allogenic mice suggest that IL-1 exerts adjuvant-like effects which increase the immunogenicity of tumor-cell antigens, and they also argue against the possibility that an IL-1-mediated local non-specific inflammatory response is the major effector mechanism of tumor rejection. Indeed, in subsequent studies we shall report on the importance of specific cellular immune responses, especially cytotoxic T lymphocytes (CTLs), in the eradication of constitutive IL-1-producing fibrosarcomas. Thus, our findings may serve as the basis for novel immunotherapy strategies aimed at the induction of IL-1 expression by cells comprising the neoplasm or alternatively by local application of the cytokine in the vicinity of the tumor.

Animals↗

PubMed1992

Interleukin-1 produced by tumorigenic fibroblasts influences tumor rejection.

Oncogene-transformed BALB/c-3T3 fibroblasts which spontaneously or upon immune-activation with cytokines and lipopolysaccharide (LPS) generate IL-1 alpha, were tested for their tumorigenicity as well as their interaction with natural immune defense by NK cells and macrophages. Oncogene-transformed fibroblasts were weakly tumorigenic, since not all mice developed tumors despite application of high doses of tumor cells. This was independent of the immune status of the host. However, in the immunocompetent host those transformed fibroblast lines which spontaneously produced IL-1 alpha grew only transiently and then regressed. After induction of IL-1 alpha production, a decrease in the rate of tumor take was noted and the rate of regression of developing tumors was increased. Regression of IL-1-producing transformed fibroblasts was strongly reduced but not completely abolished in sublethally irradiated mice. This indicated that IL-1 production may predominantly influence T-cell-mediated defense, but some influence on non-adaptive immunity could not be excluded a priori. IL-1 production did not influence susceptibility of transformed fibroblasts towards NK cells and macrophages. However, IL-1-producing transformed fibroblasts were most potent stimulators of NK cells and macrophages, the stimulatory effect being locally restricted. In conclusion, IL-1 producing, oncogene-transformed fibroblasts which generated the cytokine constitutively or upon immune-activation, were rejected from the tumor-bearing host following initial growth. Fibroblast-induced local activation of NK cells and macrophages was shown to play some role in tumor graft rejection. The influence of IL-1 production of transformed fibroblasts on T-cell-mediated defense is addressed in the accompanying report.

3T3 Cells↗

PubMed1992

Interleukin-1 production by transformed fibroblasts. II. Influence on antigen presentation and T-cell-mediated anti-tumor response.

Oncogene-transformed fibroblasts which expressed IL-1, spontaneously or after activation with conditioned medium (CM) and lipopolysaccharide (LPS), regressed in the syngeneic host. Since regression was significantly influenced by the immune competence of the host (see companion report), we speculated that regression was T-cell-mediated. Frequencies of cytotoxic T-cell precursors (CTLp) were in the same range for activated and non-activated, transformed fibroblasts. Furthermore, it was found that lysability of transformed fibroblasts was not influenced by expression of IL-1. These findings exclude the possibility that regression of CM- and LPS-treated transformed fibroblasts may have been due to the appearance of new, strongly immunogenic epitopes. On the other hand, frequencies of CTL were significantly increased after in vivo immunization with IL-1-expressing as compared to IL-1-non-expressing transformed fibroblasts. The in vivo maturation/expansion of CTL could have been the consequence of activation of helper T cells (TH), transformed fibroblast-associated IL-1 delivering the costimulatory signal. Analysis of frequencies and proliferation rates of TH confirmed this assumption. Both parameters were significantly increased after stimulation with transformed fibroblasts expressing IL-1 in comparison to transformed fibroblasts not expressing IL-1. Furthermore, purified T cells apparently depleted of cells expressing MHC class-II antigens, i.e. antigen-presenting cells, proliferated in the presence of transformed fibroblasts expressing IL-1. Since IL-1 rather than MHC class-II antigen expression was the limiting factor, antigen presentation by IL-1-expressing transformed fibroblasts appears unlikely. Instead, maturation of antigen-presenting cells could well have been initiated by tumor-associated IL-1. We conclude that IL-1 expression of transformed fibroblasts plays an important role in the induction of a T-cell-mediated anti-tumor response. The effect is due to increased efficiency in the activation of helper T cells and may be supported by activation of antigen-presenting cells.

Antigen-Presenting Cells↗

PubMed1992

Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA.

Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype.

3T3 Cells↗

PubMed1992

Establishment and characterization of monoclonal antibodies against an octahedral gallium chelate suitable for immunoscintigraphy with PET.

As a prerequisite for preparing bispecific antibody conjugates containing anti-tumor and anti-metal chelate binding sites that can be used for pretargeted immunoscintigraphy, monoclonal antibodies (Mabs) have been raised against an octahedral metal chelate synthetized from gallium (Ga) and the hexadentate ligand N,N'bis[2-hydroxy 5-(ethylene beta carboxy) benzyl] ethylenediamine N,N' diacetic acid (Ga-HBED-CC). With use of the Farr assay, binding studies with the 67Ga-labeled chelate and three clones of anti-chelate Mabs showed that none of the Mabs were able to precipitate more than 50% of the Ga-chelate, suggesting an enatiomerism of the Ga-chelate and a sensitivity of the Mabs to either one or the other chelate enantiomer. This could be confirmed by comparing the circular dichroism spectra of the Ga-chelate fractions that passed affinity columns containing the Mabs immobilized on sepharose without retention. With use of a Ga-HBED-CC enantiomer, whole-body retention in mice, preinjected with the corresponding anti-metal chelate Mab of ca. 70% ID, was measured compared to 2.1% retention in mice not preinjected with the Mab. Due to the high affinity of chelate-to-Mab binding in vivo, bispecific antibody conjugates prepared from the fragments of the anti-Ga-chelate Mab might be suitable for pretargeted immunoscintigraphy with the short-lived positron-emitter 68Ga.

Animals↗

PubMed1991

CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines.

One of several splice variants of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to the non-metastatic variant of a rat pancreatic carcinoma line (U. Günthert et al., Cell, 65: 13-24, 1991). The variant-specific rat CD44 sequences were used to detect RNA expression in human cell lines: in carcinoma lines from lung, breast and colon; and in keratinocyte lines. By polymerase chain reaction amplification, complementary DNAs encoding human homologues were isolated and sequenced. The largest splice variant has been found in a large cell lung carcinoma line and in keratinocyte cell lines. It carries at least 5 additional domains (exons) encoding a total of 338 amino acids in the membrane-proximal extracellular region of the standard CD44. Various alternative splice products have been detected in other human tumor cell lines. The distribution of CD44 splice variants is consistent with the speculation that they fulfill functions in only a few restricted differentiation pathways and that in tumor cells these pathways have been reactivated.

Amino Acid Sequence↗

PubMed1991

A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells.

Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.

Amino Acid Sequence↗

PubMed1991

Evidence for regulation of naturally activated autoreactive B cells.

A large fraction of naturally activated B cells in the neonate displays degenerate specificity, including reactivity with autoantigens. Transgenic mouse models of autoreactive B cells are mainly concerned with monospecific B cells of high avidity, and the fate of naturally activated autoreactive B cells is still a matter of debate. To pursue this question further, we chose an IgM autoantibody with a recurrent idiotype (Id), i.e. Sp6, because transgenic mice expressing this IgM also were available. In a first approach monoclonal antibodies (mAb) derived from untreated, antigenically stimulated and transgenic mice were used to test whether there were indications for deletion or for Id regulation of naturally activated autoreactive B cells. Over 90% of thymus and spleen cell derived hybridomas from 6-day-old Sp6-transgenic mice were trinitrophenyl (TNP) reactive, carried the Sp6-Id and bound to a panel of self antigens, including mouse albumin. We failed to obtain B cell hybridomas from the thymus of 28-day-old Sp6-transgenic mice. Furthermore, we could not detect any mAb carrying an anti-Sp6 Id, but Sp6 did weakly bind to itself. About 25% of mAb derived from control mice displayed degenerate specificity, the majority of them also were TNP reactive. The Sp6 Id was found at a low frequency and a comparable number of mAb carried an anti-Sp6 Id. Prenatal manipulation at the antigen level (trinitrobenzenesulfonic acid treatment) led to a transient expansion of TNP- and autoreactive mAb. The number of mAb carrying the Sp6 Id was not increased, but mAb carrying an anti-Sp6 Id were observed at high frequency. Those mAb also displayed degenerate specificity. Since Sp6-transgenic mice were perfectly healthy, it is concluded that this particular autoreactive antibody of degenerate specificity cannot be harmful for the developing organism, which may possibly be due to its self-binding capacity. Furthermore, some process of down-regulation was indicated by the absence of B cells expressing the transgene in the thymus of young adult mice. Autoreactivity of untreated and prenatally antigen-treated mice was, in addition, regulated at the Id level. In particular, mAb recognizing the Id of Sp6 were significantly expanded in antigenically stimulated mice. The data were interpreted in the sense that autoreactive B cells appearing early during ontogeny were rather strictly controlled either by (functional) clonal deletion or by idiotypic connectivity.

Animals↗