Biomedical subjects

R Zeidler

Publications and source records attributed to R Zeidler.

At least 19 recordsLinked to original sources

PubMed2006

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells.

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.

B-Lymphocytes↗

PubMed2004

Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology.

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Allergy and Immunology↗

PubMed2002

[Giant Madelung's disease. Report of a case and review of the literature].

Madelung's disease, also known as benign symmetrical lipomatosis, is a rare proliferative disorder of unknown etiology that was first mentioned by Brodie in 1846. Characterized by multiple symmetrical deposits of unencapsulated fat in the head and neck region, the disease is most common in middle-aged men with a history of alcohol abuse. The only effective therapy in cases of dyspnea and dysphagia, indicating the necessity of treatment, is the surgical resection of the adipose tissue. The authors report on the evaluation and therapy in a case of giant Madelung's disease.

Adipose Tissue↗

PubMed2001

Tumor necrosis factor alpha negatively regulates the expression of the carcinoma-associated antigen epithelial cell adhesion molecule.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is a homophilic and Ca2+ independent adhesion molecule that is expressed de novo in squamous cell carcinoma (SCC) but is absent in the majority of healthy squamous epithelia. EpCAM expression correlates with cell proliferation and dedifferentiation along with a progression in tumorigenicity. To date, nothing is known about the molecular mechanisms responsible for the regulation of the EpCAM gene. METHODS: The authors analyzed the regulation of a fragment of the EpCAM promoter. RESULTS: The analyzed fragment has significant activity in EpCAM positive cells, and it is regulated negatively by tumor necrosis factor alpha (TNFalpha). This negative regulation results in diminished mRNA expression and in the down-regulation of EpCAM protein at the cell surface in SCC cells. Both effects can be mimicked by the treatment of cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). TNFalpha-induced inhibition of the EpCAM expression is mediated by TNF receptor 1 through the TNF receptor-associated death domain protein (TRADD) and by the activation of nuclear factor kappaB (NF-kappaB), and it can be blocked by dominant-negative variants of TRADD and the NF-kappaB inhibitor, IkappaB. The authors provide further evidence that NF-kappaB represses EpCAM expression by competing for the transcriptional coactivator p300/CREB binding protein (p300/CBP). CONCLUSIONS: The current results provide the first insights into the regulation of EpCAM expression, which is regulated negatively by TNFalpha and TPA through the activation of NF-kappaB. The repression may rely on the competition of NF-kappaB for p300/CBP histone acetyl transferase activity, because the overexpression of p300 reverts TNFalpha effects.

Antigens, Neoplasm↗

PubMed2001

[IL-2 gene therapy in ENT carcinomas].

Suppressed cellular immunity is common in patients with squamous cell carcinoma of the head and neck (HNSCC). It was demonstrated in previous studies that administration of interleukin 2 (IL-2) results in enhanced antitumoral immunity in vitro as well as in vivo. Since the serum half-life of IL-2 is relatively short, repeated applications are necessary to achieve therapeutically effective serum concentrations, but this strategy might cause severe side effects. Therefore, methods that provide high local cytokine levels over a prolonged period of time without the need for repeated injections are desirable. Gene therapy as an innovative treatment approach using tumor cells stably transduced to produce IL-2 might meet these criteria. In vitro manipulated tumor cells, if readministered in the vicinity of non-manipulated tumor cells, may enhance a specific anti-tumor response in vivo without systemic side effects. The present manuscript reviews the current literature dealing with IL-2-protein and -gene therapy with special emphasis on head and neck cancer. Our own in vitro results with IL-2 gene therapy in conjunction with published data from other authors argue in favour of an in vivo approach for this therapeutic strategy that is currently in progress in our department.

Animals↗

PubMed2000

The PKC targeting protein RACK1 interacts with the Epstein-Barr virus activator protein BZLF1.

Phorbol esters reactivate Epstein-Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate-early gene BZLF1. BZLF1 is a member of the extended AP-1 family of transcription factors that binds to specific BZLF1-binding motifs within early EBV promoters and to consensus AP-1 sites. Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post-translational modifications. Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA [Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J. & Hammerschmidt, W., (1998) J. Virol. 72, 8105-8114]. The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay. RACK stands for receptor of activated C-kinase and is involved in targeting activated PKCs and other signaling proteins. In vivo, RACK1 binds directly to the transactivation domain of BZLF1. Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays. We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.

Amino Acid Sequence↗

PubMed2000

The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells.

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 x anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcgamma-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUII-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUII to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcgamma-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis.

Antibodies, Bispecific↗

PubMed2000

Tumor cell-derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac-1.

The cyclooxygenases (COX)-1 and COX-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Whereas COX-1 is expressed ubiquitously, COX-2 is an immediate-early gene often associated with malignant transformation, and a role for the COX enzymes in tumor initiation and promotion is discussed. Nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin and indomethacin that block COX-1 and -2 have been shown to have beneficial effects for tumor patients. Therefore, these compounds have gained interest also among oncologists. However, the molecular mechanism by which NSAIDs inhibit carcinogenesis is not clearly understood. The prostaglandin-dependent and -independent effect may both account for their antineoplastic action. We show here that tumor cells derived from different tumors regularly produce prostaglandin E(2) (PGE(2)) interfering with the function of monocytes. In particular, PGE(2) inhibits the potential of monocytes to migrate in the direction of a chemotactic stimulus and to adhere to endothelial cell. This inhibition is most probably due to a modulation of the chemokine receptor CCR5 and the beta2-integrin Mac-1. Both down-regulation of CCR5 and reduced expression of Mac-1 may diminish the potential of peripheral blood monocytes to leave blood vessels and invade target tissues. Since both dysfunctions can be restored with NSAIDs, our findings help to explain the molecular chemopreventive action of NSAIDs on tumor formation and progression.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing.

Bispecific Abs (bsAb) are promising immunological tools for the elimination of tumor cells in minimal residual disease situations. In principle, they target an Ag on tumor cells and recruit one class of effector cell. Because immune reactions in vivo are more complex and are mediated by different classes of effector cell, we argue that conventional bsAb might not yield optimal immune responses at the tumor site. We therefore constructed a bsAb that combines the two potent effector subclasses mouse IgG2a and rat IgG2b. This bispecific molecule not only recruits T cells via its one binding arm, but simultaneously activates FcgammaR+ accessory cells via its Fc region. We demonstrate here that the activation of both T lymphocytes and accessory cells leads to production of immunomodulating cytokines like IL-1beta, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb elicits excellent antitumor activity in vitro even without the addition of exogenous IL-2, and therefore represents a totally self-supporting system.

Animals↗

PubMed1999

Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.

Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein-Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF-kappaB and AP-1 via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1's C-terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF-R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

Amino Acid Sequence↗

PubMed1999

Gene therapy--phase I trial for primary untreated head and neck squamous cell cancer (HNSCC) UICC stage II-IV with a single intratumoral injection of hIL-2 plasmids formulated in DOTMA/Chol.

Recombinant IL-2 protein has shown many immunostimulatory effects in a variety of human tumors. However, the clinical use of rIL-2 is limited by common and serious side effects after systemic administration. IL-2 expression plasmids may circumvent these drawbacks, producing high local IL-2 concentrations that cause limited or no systemic side effects. Due to the superficial growth of squamous cell carcinoma of the head and neck (HNSCC) are readily accessible for direct intratumoral injection and therefore an optimal target for such a gene therapy approach. There has been evidence for local and systemic activation of immune cells by peritumoral injections of IL-2 in patients with advanced HNSCC (Whiteside et al. 1993; Cortesina et al. 1994; De Stefani et al. 1996). We now perform a placebo-controlled, dose-rising study of the safety and tolerability of a single intratumoral injection of hIL-2 plasmid at four dose levels formulated in DOTMA/Chol in patients with primary untreated head and neck squamous cell cancer (HNSCC) TNM stage II-IV. The patients will be monitored for the occurrence of any adverse reactions to the given medication. In addition, we will determine whether the intratumoral administration of the plasmid induces and or enhances tumor-specific host responses at the immunological and or clinical level.

Carcinoma, Squamous Cell↗

PubMed1999

Impairment of T-cell activation in head and neck cancer in situ and in vitro: strategies for an immune restoration.

BACKGROUND: The rationale for the study was based on the hypothesis that decreased or absent expression on tumor cells of adhesion molecules, the class I or class II major histocompatibility complex (MHC) molecules, or costimulatory molecules might be responsible, in part, for the poor ability of squamous cell carcinoma of the head and neck (SCCHN) to induce generation of antitumor effector cells in vitro and in vivo. OBJECTIVE: To investigate expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-3 (LFA-3) and distribution of the costimulatory molecules, B7.1, B7.2, and CD40, and of class I and class II MHC molecules on SCCHN cells in situ and on SCCHN cell lines. SETTING: University medical centers. DESIGN: Expression of ICAM-1, LFA-3, MHC molecules, B7.1, B7.2, and CD40 was evaluated in human SCCHN biopsy specimens by immunohistochemistry and on SCCHN cell lines by flow cytometry. To confirm our hypothesis that impaired T-cell activation observed in patients with SCCHN is caused by the absence of costimulatory B7 molecules, a B7-negative SCCHN cell line was transduced with the B7.1 gene, using a retroviral vector, and tested in mixed lymphocyte tumor cocultures. RESULTS: In contrast to abundant expression of ICAM-1, LFA-3, class I MHC molecules, and CD40, the absence of B7.1, B7.2, and class II MHC molecules on tumor cells was observed in situ and in vitro. Lymphocytes and antigen-presenting cells in inflammatory infiltrates surrounding tumor cell clusters expressed both costimulatory and adhesion molecules. The SCCHN lines negative for B7.1 and class II MHC antigens failed to induce proliferation of T cells in mixed lymphocyte tumor cocultures. However, when these cell lines were transduced with the B7.1 gene, their ability to induce T-cell proliferation in mixed lymphocyte tumor cocultures was restored. CONCLUSIONS: The absence of B7 protein or class II MHC antigen expression on human SCCHN cells is responsible for the failure of these tumors to induce proliferation of T cells in vitro. Transduction of the B7.1 gene into SCCHN restores the ability of the tumor to induce T-cell proliferation in vitro.

Adult↗

PubMed1999

[Induction of antitumor immune response in the mouse model after vaccination with B7.1 expressing tumor cells].

The aim of the study was the induction of an antitumor immune response by genetic modification of tumor cells. This was done by transfecting the costimulatory molecule B7.1 into a murine tumor cell line SCCVII/SF in order to increase T cell recognition and to install an immunologic memory. One cohort of immunocompetent mice C3 H/HeN were injected with B7.1 expressing tumor cells, while the control group received parental B7.1 negative tumor cells. In a second step those immunized mice were rechallenged by parental tumor cells and tumor growth was compared to a new control group. Transfection with B7.1 prohibits outgrowth of the SCCVII cell line. Animals that have been vaccinated in this way are partially immune towards a secondary exposition to B7.1 negative tumor cells. Outgrowth of this recurrent tumor is slowed down. Such an immunization builds up an immunologic memory. Vaccination with B7.1 expressing tumor cells lead to a partial protective tumor immunity in the SCCVII-C3 H/HeN mouse model.

Animals↗

PubMed1998

Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells.

With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.

B-Lymphocytes↗

PubMed1998

Activation of the Epstein-Barr virus transcription factor BZLF1 by 12-O-tetradecanoylphorbol-13-acetate-induced phosphorylation.

BZLF1 is a member of the extended AP-1 family of transcription factors which binds to specific BZLF1 sequence motifs within early Epstein-Barr virus (EBV) promoters and to closely related AP-1 motifs. BZLF1's activity is regulated at the transcriptional level as well as through protein interactions and posttranslational modifications. Phorbol esters or immunoglobulin cross-linking both reactivate EBV from latently infected B cells via transactivation of BZLF1. We report here that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is capable of inducing BZLF1's activity even further. The induction occurs at the posttranscriptional level and depends on a single serine residue located in the DNA binding domain of BZLF1. This serine residue (S186) is phosphorylated by protein kinase C in vitro and in vivo after stimulation with TPA. Phosphorylation of S186 per se interferes with the DNA binding affinity of BZLF1 in vitro but is mandatory for TPA-induced increase in DNA binding of BZLF1, as shown in gel retardation assays and reconstruction experiments with cellular extracts. In transcriptional reporter assays, S186 is essential for the activation of BZLF1 by TPA. Presumably, a yet-to-be-identified cellular factor restores the DNA binding affinity and enhances the transcriptional activity of S186-phosphorylated BZLF1, which is required to induce the lytic phase of EBV's life cycle.

Amino Acid Sequence↗

PubMed1998

Lack of B7.1 and B7.2 on head and neck cancer cells and possible significance for gene therapy.

Cellular immunity mediated by cytotoxic T-cells plays a key role in host tumor defense. An optimal T-cell recognition is based primarily on the presentation of an antigen in the context with MHC Class I molecules, secondarily co-stimulatory molecules, such as the B7 family, are necessary to initiate maximum stimulation. We examined the quality (immunohistochemically) and quantity (FACS-analysis) of B7 expression on primary and permanently established head and neck squamous cell cancer (HNSCC). Neither on the primary nor on the permanently established HNSCC lines could B7 be detected. The lack of co-stimulatory molecules could be the reason for the low immunogenicity of HNSCC. Future gene transfer of B7 could help to restore the immune function. Transfection of expression plasmids encoding B7 cDNA into the tumor cells by gene therapy might restore tumor-specific immunity as a new tool for cancer eradication in the future.

Journal Article↗

PubMed1997

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.

B-Lymphocytes↗

PubMed1997

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗