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Biomedical subjects

G Moldenhauer

Publications and source records attributed to G Moldenhauer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Cell surface sialylation plays a role in modulating sensitivity towards APO-1-mediated apoptotic cell death.

APO-1/Fas(CD95), a member of the tumour necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily transduces apoptotic signals into apoptosis sensitive cells. In metabolic labelling experiments using the highly APO-1 positive cell lines HUT78 (adultT cell leukemia) and SKW6.4 (Blymphoblastoid cell line) APO-1 was characterised as a long living protein with a complex glycosylation pattern involving terminal sialic acid groups which account for 8-kDa of its apparent molecular weight on SDS-PAGE. APO-1 expression and the degree of sialylation were determined in additionalT and B cell lines. On the group I Burkitt's lymphoma cell line BL60 transfected with human APO-1 (K50) low sialylated species were detected only on the cell surface, suggesting that sialylation might be functionally important. Removal of terminal sialic acid groups by treatment of B and T cell lines with Vibrio cholerae neuraminidase (VCN) augmented sensitivity towards anti-APO-1 and human APO-1 ligand induced apoptosis. Similarly, VCN-treated U937 cells were rendered more sensitive to TNFalpha-induced cell death. Thus, sialylation may be one mechanism to regulate sensitivity towards ligand-mediated cell death in this receptor family.

Journal Article↗
PubMed1994

Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.

To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.

Amino Acid Sequence↗
PubMed1994

Lymphotoxin-alpha/beta heterodimer is expressed on leukemic hairy cells and activated human B lymphocytes.

The expression of human lymphotoxin (LT) alpha/beta cell-surface complex was studied in human B-cell lines as well as in normal and neoplastic human B lymphocytes. In the absence of TNF receptors, only the human hairy-cell leukemia (HCL)-derived cell line JOK-I revealed constitutive cell-surface expression of LT but not TNF-alpha. Immunoprecipitation experiments with anti-LT monoclonal antibody (MAb) 9B9 from cell-surface radioiodinated JOK-I cells revealed that a cell-surface lymphotoxin molecule (25 kDa) is expressed in association with a 33-kDa molecule. Enzymatic digestion with F/N-glycosidase and O-glycosidase showed that both proteins contained N-linked carbohydrate residues, whereas only the 25-kDa molecule contained O-linked sugar residues. Analysis of mRNA expression revealed specific transcripts of LT-alpha and LT-beta in JOK-I cells. Resting tonsillar B cells did not express cell-surface LT. However, LT-beta mRNA was observable in unstimulated tonsillar B cells, whereas LT-alpha mRNA, cell-surface LT and LT secretion could only be detected upon in vitro activation. Thus LT-beta and alpha appear to be sequentially expressed in human B cells. Neoplastic B cells from chronic lymphocytic leukemia (BCLL), being devoid of constitutive cell-surface LT expression, could be induced to express surface LT by in vitro stimulation with Staphylococcus aureus Cowan I (SAC). Constitutive LT-beta transcripts, however, could also be detected in 4 out of 5 cases of BCLL. In contrast, human HCL cells displayed constitutive cell expression of lymphotoxin-alpha and beta. These findings demonstrate that cell-surface LT-alpha is expressed in association with LT-beta on activated normal B cells and neoplastic B cells representing an activated state.

B-Lymphocytes↗
PubMed1993

Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies.

Bispecific monoclonal antibodies can be used in the activation of effector cells to lyse autologous tumor cells. We analyzed the activation of human T-cells in vitro with bispecific monoclonal antibodies, which were generated by hybridoma-hybridoma fusion. Preactivated allogeneic and autologous T-cells could be triggered to lyse tumoral B-cells in the presence of CD3 x CD19 bispecific antibodies. In addition, the combined use of two CD3 x CD19 plus CD28 x CD22 bispecific antibodies induced optimal interleukin 2 secretion by Jurkat T-cell acute lymphocytic leukemia cells in the presence of target B-cells. The same antibody combination was able to generate cytolytic effector cells without prior activation, when resting T-cells were cocultured with freshly isolated autologous leukemic B-cells in the presence of the bispecific antibodies. The results suggest that signals required to activate cytolytic T-cell precursors can be provided by the two bispecific antibodies. Although activation of resting T-cells can be achieved by CD3 x CD19 bispecific antibodies in association with monospecific bivalent CD28 antibodies, the second bispecific antibody, CD28 x CD22, further increases the specificity of the target cell dependent activation of T-cells. When used for immunotherapy of B-cell malignancies, the CD3 x CD19 and CD28 x CD22 bispecific antibody combination may avoid the need for ex vivo activated effector cells, because the antibodies may induce T-cell activation directly at the tumor site.

Animals↗
PubMed1993

Monoclonal antibody uptake in B-cell lymphomas: experimental studies in nude mouse xenografts.

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.

Animals↗
PubMed1992

Separation of tumor cells from a suspension of dissociated human colorectal carcinoma tissue by means of monoclonal antibody-coated magnetic beads.

Dissociation of colorectal tumor tissue directly obtained at surgery yields a cell suspension which contains tumor cells, leukocytes and erythrocytes. Coupling of a murine monoclonal antibody, HEA125, specific for human epithelial cells, to magnetic beads permits positive selection of a population containing essentially only tumor cells. Removal of cells from the beads is possible by papain treatment. Negative selection of tumor cells by means of beads coated with a leukocyte binding antibody offers no advantage since dead cells and erythrocytes remain.

Adenocarcinoma↗
PubMed1992

Local growth of a Burkitt's lymphoma versus disseminated invasive growth of the autologous EBV-immortalized lymphoblastoid cells and their somatic cell hybrids in SCID mice.

Specific host-graft interactions, as well as intrinsic properties of transferred cell, determine tumorigenicity in xenogeneic systems. We compared the growth characteristics of human B-lymphoid cell lines in SCID mice with the well characterized growth pattern in nude mice and observed striking differences in malignancy in the respective hosts. Two cell lines derived from the same individual, the Epstein-Barr-virus(EBV)-positive Burkitt's lymphoma BL 60 (BL) and the autologous EBV-immortalized lymphoblastoid cell line IARC 277 (LCL) were used. In addition, we tested somatic cell hybrids (HYB) of both cell lines, which despite the LCL-like differentiation phenotype show the de-regulated c-myc expression pattern of the parental BL line, assumed to be a critical factor in BL pathogenesis. Subcutaneously (s.c.) injected BL cells produced local progressively growing tumor masses at the injection site without distant metastases in both nude and SCID mice. Although both mouse strains possess the same genetic background (BALB/c) and differ only in the B-cell sub-set, the growth patterns of the LCL and hybrids were completely different. In contrast to the regressive behaviour of LCL and hybrids in nude mice, these lines show invasive and disseminated progressive growth in SCID mice. Peripheral lymph nodes an thymic tissue were preferentially colonized, whereas mucosal-associated lymphoid tissue (Peyer's patches and appendix) and spleen were not infiltrated. The preferential migration of lymphocytes to certain tissues is termed homing in a syngeneic system and mediated by homing receptors and vascular addressins. The "homing" of LCL and hybrids into lymphoid SCID mouse tissue suggests a strong interaction with the endothelial cells of the host. Detailed phenotypic analysis of BL, LCL and 3 different hybrids was performed using an antibody panel against differentiation and adhesion markers. Overall dominance of the LCL phenotype was observed in the hybrids, as indicated by cytology, tumor growth, dissemination and the pattern of surface-marker expression. The c-myc activation in hybrids does not appear to influence growth behavior.

Animals↗
PubMed1992

Monoclonal antibodies to idiotype inhibit in vitro growth of human B-cell lymphomas.

The effect of monoclonal antibodies (MoAbs) recognizing idiotype, IgM heavy chain, and IgD heavy chain on the in vitro DNA synthesis of five randomly selected leukemic human low-malignancy B-cell lymphomas was investigated. In three lymphomas of different histologic subtype, low concentrations of anti-idiotypic (anti-Id) MoAb completely inhibited spontaneous 3H-thymidine uptake of T-cell-- and monocyte-depleted tumor cells, whereas two other tumors were not affected. Maximal inhibition of DNA synthesis was achieved at MoAb concentrations ranging from 0.5 to 250 micrograms/mL and required crosslinking by bivalent antibody but not Fc-mediated effects. While two anti-IgM MoAbs were similarly efficient as anti-Id MoAb in inhibition of DNA synthesis, two anti-IgD MoAbs had no effect. Thus, surface IgD molecules seemed to be neither able to deliver inhibitory signals themselves nor to antagonize IgM-mediated signals when simultaneously crosslinked by anti-Id MoAb. Leukocyte differentiation antigen expression, IgM density, and IgM/IgD ratio on the surface of lymphoma cells did not distinguish between sensitive and resistant tumors. In vitro tumor cell survival was differently affected by prolonged incubation with anti-Id antibody. In a centrocytic lymphoma and an immunocytoma, but not in a chronic lymphocytic leukemia, suppression of 3H-thymidine uptake persisted after removal of MoAb and tumor cell viability decreased during prolonged incubation with anti-Id MoAb. These results suggest that direct inhibitory signaling via surface IgM may contribute to anti-Id MoAb-mediated tumor regression in certain human B-cell lymphomas.

Antibodies, Monoclonal↗
PubMed1992

Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias.

S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.

Antibodies, Monoclonal↗
PubMed1992

Inhibitory and stimulatory signaling via immunoglobulin receptors: dichotomous responses elicited in clonal B cell populations.

The paradox that cross-linking of IgM antigen receptors on B cells by native or surrogate antigen can inhibit, as well as stimulate, B cell functions has previously been attributed to changes during maturation and activation, programming either a negative or a positive response at defined developmental stages. In contrast to this concept, we show here that some B cells possess the potential for both types of response at the same stage. In three clonal malignant human B cell populations, bivalent soluble monoclonal antibodies to IgM or idiotype, but not IgD completely inhibited spontaneous DNA synthesis, but significantly induced [3H]thymidine uptake when coupled to insoluble compounds. In co-incubation experiments mitogenic stimuli were dominant over inhibitory ones and were still effective after prolonged pretreatment of the B cells with inhibitory reagents. Ionomycin, known to increase intracellular calcium levels, and low doses of phorbol ester, described to activate protein kinase C, also suppressed DNA synthesis. High doses of phorbol ester alone or in combination with ionomycin, however, induced DNA synthesis in two of the lymphomas. We conclude that some B cells may respond to cross-linking of surface IgM in a dose-dependent manner so that all signals increase DNA synthesis once a threshold has been reached. These dose-dependent effects may in part involve signaling via breakdown of membrane inositol phosphates.

Antibodies, Monoclonal↗
PubMed1992

Human Golgi beta-galactoside alpha-2,6-sialyltransferase generates a group of sialylated B lymphocyte differentiation antigens.

The role of the human beta-galactoside alpha-2,6-sialyltransferase (hu alpha-2,6-ST) in the generation of B cell surface antigens was investigated by selecting subclones of COS cells (monkey kidney epithelial cells) constitutively expressing a transfected cDNA which encodes the hu alpha-2,6-ST (COS alpha-2,6-ST cells). Expression of hu alpha-2,6-ST in COS cells was sufficient to generate sialylated cell surface epitopes on different glycosylated antigens recognized by monoclonal antibodies to CDw75, CD76, and the unclustered monoclonal antibodies HB-4 and EBU-65. These epitopes were sensitive to sialidase treatment and are likely to contain terminal alpha-2,6-linked sialic acid residues. A novel antiserum raised against bacterially expressed hu alpha-2,6-ST fusion protein was used to localize the sialyltransferase in two cell lines with high expression of either endogenous (B cell line JOK-1) or recombinant (COS alpha-2,6-ST cells) hu alpha-2,6-ST. In both cell lines, the enzyme was detected only intracellularly in the juxtanuclear region and not on the cell surface. In contrast, CDw75, formerly proposed to be identical with an alpha-2,6-ectosialyltransferase, was strongly expressed on the cell surface. The different expression patterns show that neither the CDw75 antigen nor any of the other sialylated antigens analyzed is identical with the hu alpha-2,6-ST. Furthermore, the presence of a surface-expressed alpha-2,6-ST appears unlikely in these cell lines. We propose that CDw75, CD76, HB-4, and EBU-65 represent a unique group of B cell differentiation antigens the production of which requires the enzymatic activity of alpha-2,6-ST.

Animals↗
PubMed1992

Three types of human lung tumour cell lines can be distinguished according to surface expression of endogenous urokinase and their capacity to bind exogenous urokinase.

This study evaluates the cell surface expression of urokinase-type plasminogen activator (u-PA) and the capacity to bind exogenous urokinase as possible parameters for the distinction of various types of human lung tumours. Twelve different tumour cell lines including four small cell carcinoma, two large cell carcinoma, three squamous cell carcinoma, one adenocarcinoma and two mesothelioma cell lines of lung origin were investigated. Surface expression of endogenous u-PA was determined in a cellular radioimmunoassay (CRIA) using the u-PA-specific monoclonal antibody 98/6. To estimate additional u-PA binding capacity, exogenous two-chain, 54 kDa u-PA was employed in the CRIA. The influence of phorbol ester (PMA) treatment on expression and binding of these molecules was studied. Three different groups of lung tumour cell lines could be distinguished according to their expression of u-PA and u-PA-binding ability: (i) non small cell lung carcinoma (NSCLC) cell lines of squamous cell carcinoma/adenocarcinoma origin expressed small amounts of u-PA and bound little u-PA. Large cell carcinoma cell lines expressed high amounts of u-PA and bound large amounts of u-PA. In general, expression of u-PA and u-PA binding was enhanced after PMA treatment. (ii) Mesothelioma cell lines did not express u-PA, but were able to bind u-PA. (iii) Small cell carcinoma (SCLC) lines were devoid of surface-expressed u-PA and could not bind u-PA, both under untreated and PMA-treated conditions. It could thus be demonstrated that these three groups of lung tumour cell lines differ in their ability to express u-PA and to bind external u-PA. This may reflect the different in vivo growth behaviour and origin of the respective tumour groups.

Adenocarcinoma↗
PubMed1992

Multiple ICAM-1 (CD54) epitopes are involved in homotypic B-cell adhesion.

Two monoclonal antibodies (MoAbs), F10.2 and F10.3, were selected for their ability to interfere in homotypic adhesion of human B cells. Precipitation studies and binding to intercellular adhesion molecule 1 (ICAM-1, CD54) cDNA transfected COS cells revealed that both MoAbs are directed against ICAM-1. The binding of MoAb F10.2 was inhibited by LB-2, a MoAb recognizing the NH2-terminal immunoglobulin-like domain of ICAM-1. This suggests that the epitope recognized by F10.2 is located on the first domain of the ICAM-1 molecule. Binding of the other MoAb, F10.3, was not inhibited by F10.2 nor by two other MoAbs mapping to the first domain of the ICAM-1 molecule. The ability of F10.3 to bind to ICAM-1 is influenced by glycosylation, suggesting that this epitope is located on one of the domains carrying possible glycosylation sites, i.e. domain 2, 3 or 4. The ICAM-1 epitopes recognized by F10.3 and LB-2 or F10.2 co-operated in homotypic adhesion of cells from the EBV cell line ML1. These results suggest that in addition to an epitope located on domain 1 of the ICAM-1 molecule, another epitope whose exposure can be regulated by glycosylation is involved in homotypic B-cell adhesion of cell line ML1.

Antibodies, Monoclonal↗
PubMed1991

Rapid intracellular pathway gives rise to cell surface expression of the MHC class II-associated invariant chain (CD74).

In previous investigations, it had been shown that class II and associated invariant polypeptides are sorted to an endocytic route where transport is delayed. Invariant chain (Ii) is degraded in a post-Golgi compartment, presumably an endosomal vesicle, and only class II molecules emerge on the cell surface. By using a mAb against the extracytoplasmic domain of human Ii, we demonstrate, by electron microscopy and by cytofluorometry, surface expression of Ii on lymphoma cells and on human B lymphocytes. We examined surface expression of Ii upon inhibition by brefeldin A of intracellular transport from the endoplasmic reticulum to the Golgi stack. This treatment rapidly depletes the cell surface of Ii. In the subsequent absence of brefeldin A, Ii appears rapidly at the cell surface. Within 5 h, the previous level of surface Ii (sIi) is reconstituted. Chloroquine abrogates depletion of sIi by brefeldin A, apparently by inhibition of internalization of sIi. Because on its route to the cell surface Ii is not proteolytically digested, it was possible that Ii and associated class II molecules are not separated on this pathway. Immunochemical studies reveal that on the cell surface of a B lymphoma cell line a proportion of Ii and class II polypeptides are associated.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1991

Influence of major histocompatibility complex class I and II antigens on survival in colorectal carcinoma.

HLA-A,B,C and HLA-D molecules present antigenic peptides to the antigen-specific receptor of autologous T-lymphocytes. T-cell-mediated host-versus-tumor response might therefore depend on the presence of these molecules on tumor cells, although the absence of HLA-A,B,C determinants on a cell has been shown to increase its susceptibility to lysis by natural killer cells. To investigate whether the presence or absence of HLA-A,B,C and/or HLA-DR in colorectal carcinoma influences relapse rate and time of tumor-related death, 152 patients who underwent putatively curative surgical treatment were surveyed for a maximum of 65 months (mean, 48 months). As determined by immunohistochemistry, aberrant reduction or loss of HLA-A,B,C/beta 2-microglobulin molecules was more frequent in tumors of the proximal colon than of the rectosigmoid (P = 0.032) and in mucinous carcinomas than in nonmucinous ones (P = 0.022). An abnormal induction of the HLA-D-associated invariant chain (Ii) was more frequent in Dukes' A and B than in stage C (P = 0.046). Reduction/loss of HLA-A,B,C/beta 2-microglobulin was correlated with the absence of HLA-DR (P = 0.024) and Ii (P = 0.005). In contrast to the prognostic role of tumor stage and grade, the presence versus the absence of HLA-A,B,C/beta 2-microglobulin and HLA-DR/Ii molecules was not correlated with recurrence rate or survival. We conclude that in spite of an increasing amount of experimental data suggesting the contrary, the status of HLA-A,B,C and HLA-DR expression in colorectal carcinoma seems to be irrelevant in vivo, regarding survival and growth of residual tumor cells after putatively curative resection of the initial tumor burden.

Aged↗
PubMed1991

Regressing nude mouse grafts of Burkitt's lymphoma x lymphoblastoid cell hybrids show deregulation of the c-myc gene and expression of the EBV latent membrane protein.

Somatic cell hybrids between the malignant Burkitt lymphoma cell line BL 60 and the non-malignant Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) IARC 277 demonstrate the deregulated c-myc transcription pattern of the parental BL cell line during exponential growth in tissue culture. Subcutaneous nude mouse grafts of these hybrids, however, completely regress after an initial growth phase. To investigate whether regression of these grafts is mediated by a down-regulation of the BL-60-derived deregulated c-myc gene in vivo, c-myc transcription was analyzed in growing versus regressing hybrid grafts. In the initial growth phase as well as during regression, these grafts showed the deregulated c-myc expression pattern of the parental BL cell line with highly abundant c-myc transcripts originating from the BL specific translocation chromosome 8q+. Our results question the significance of c-myc deregulation for an unlimited in vivo growth potential of B-lymphoblastoid cells. To further characterize the hybrids, surface expression of B-cell-specific antigens was analyzed on the hybrids and shown to correspond to that of the parental LCL. In addition, transcription of the gene encoding for the EBV latent membrane protein (LMP) as well as histological features were analyzed in growing versus regressing hybrid grafts. The LMP gene, which is down-regulated in the tumors produced by the parental BL cells, was expressed in the hybrid grafts as well as in the parental LCL grafts. This finding might be compatible with a host response of the nude mouse against LMP. The histological analysis, however, which revealed massive necrosis in the center of the regressing grafts without pronounced inflammatory infiltrates, rather points to a hypoxemic process leading to graft regression.

Animals↗
PubMed1991

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.

Antibodies, Monoclonal↗