Biomedical subjects

J Zeuthen

Publications and source records attributed to J Zeuthen.

At least 55 records · Page 3Linked to original sources

PubMed1998

Sticky and smelly issues: lessons on tumour cell and leucocyte trafficking, gene and immunotherapy of cancer.

The Second Meeting of the British Society for Immunology Tumour Immunology Affinity Group (TIAG) took place at King's College (London, UK) on 17-18 June 1997 and brought together over 100 tumour immunologists from the UK and abroad. In contrast to previous meetings the focus of the meeting was on the role of adhesion in immunosurveillance and tumour dissemination. In addition, recent achievements in the areas of chemokines, cytotoxic T-lymphocyte (CTL) and natural killer (NK) cells, co-stimulation, gene and adoptive immunotherapy were also addressed. The purpose of this report is to outline current trends in tumour immunology.

Animals↗

PubMed1998

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb.

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.

Adult↗

PubMed1998

T-cell receptor variable region genes in cutaneous T-cell lymphomas.

The genes encoding the T-cell receptor (TCR) variable beta (TCRBV) regions were studied in skin biopsy samples from 24 patients with cutaneous T-cell lymphomas (CTCL), i.e. mycosis fungoides (n = 7), Sézary syndrome (n = 4), lymphomatoid papulosis (n = 3) and large cell CTCL with (n = 3) or without (n = 7) CD30 expression. A panel of 24 primers specific for TCRBV families 1-24 was designed and applied to cDNA using a semiquantitative reverse transcriptase coupled polymerase chain reaction (PCR) method. Three patients showed restricted expression of a limited number (1-3) of TCRBV families. In the remaining patients, an average of 17 (range: 13-22) different families was expressed. All patients showed elevated (> 10%) expression of individual families significantly higher than that seen in normal blood lymphocytes, but no preferential usage of particular gene families was observed. Direct sequence analysis of more than 60 PCR products revealed clonal TCR transcripts in 18 patients. A single T-cell clone, constituting 9-100% (mean: 26%) of the TCRBV mRNA, was present in 12 patients; two T-cell clones, constituting 13-72% (mean: 21.5%) of the TCRBV mRNA, were present in five patients; and three T-cell clones, accounting for < 0.5-13% of the TCRBV mRNA, were present in one patient. In the remaining six patients, clonal TCR transcripts could not be identified. It is concluded that most CTCL contain both clonal and non-clonal (reactive) T cells, and that the latter cells are more numerous than anticipated. These cells may be engaged in tumour immune reactions, although prospective studies and/or serial investigations are needed to elucidate this issue fully.

Adult↗

PubMed1998

Melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last 7 years significant progress has been made in the identification of melanoma-associated antigens recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: cancer/testis-specific antigens (MAGE, BAGE, GAGE, PRAME and NY-ESO-1), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2), and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). In this review we have summarized the available data concerning the characterization of melanoma-associated antigens, focusing on their immunogenic and protective properties. The development of a strong immune response to differentiation antigens is limited by the existence of tolerance to these "self"-antigens, permitting the involvement of only T cells with low affinity T-cell receptors. Among the melanoma differentiation antigens, only gp100 has been shown to be a tumor regression antigen. The cancer/testis-specific antigens such as MAGE and PRAME should potentially be highly immunogenic antigens. They contain several potential HLA class I binding epitopes and are present only in the testes, which are not accessible to the cells of the immune system owing to the lack of direct contact with the immune cells and the lack of HLA class I expression on the surface of germ cells. But only two patients have been found who responded to these antigens in vivo, indicating their genuinely low immunogenicity. A comparison of the predicted secondary structures of these two groups of antigens (cancer/testis-specific and differentiation antigens) revealed enrichment of long alpha-helical stretches in the cancer/testis-specific antigens. We hypothesize that such highly organized stable structures could, first, reduce denaturation of the protein and, thus, ubiquitinylation as a degradation signal, and, second, diminish the efficiency of the protein unfolding - a necessary step in the proteolytic cleavage by proteasomes. High structural stability could therefore be responsible for the low immunogenicity of these proteins. In this case, modifications decreasing the stability of these proteins might be a means of improving the immune response to these potentially therapeutically useful antigens.

Amino Acid Sequence↗

PubMed1998

A comparison of flow cytometry and immunohistochemistry in human colorectal cancers.

In human colorectal cancer it has been reported that some tumours lack the HLA-ABC antigens. This has been interpreted as reflecting tumour escape from the immune system. Earlier data have been obtained by immunohistochemistry. In this study, we compared the expression of HLA-ABC, HLA-DR, CD80 (B7-1) and CD54 (ICAM-1) in 20 tumours using both a conventional immunohistochemistry two-layer technique and multiparameter flow cytometry, gating on an epithelial cell marker. Colorectal cancer tissue used in flow cytometry was dissociated with collagenase, deoxyribonuclease and hyaluronidase. The intensity of expression of HLA-ABC, HLA-DR and CD80 was unaffected by the enzymes, but CD54 was decreased by 30%. The reproducibility of flow cytometry was good. Microscopy of sections revealed that about 5% of each tumour sample consisted of normal epithelium, but even after correction for this, flow cytometry was superior to immunohistochemistry in 33 out of 80 cases, and showed that tumours described as HLA-ABC negative by immunohistochemistry were in fact weakly positive for HLA-ABC. We conclude that flow cytometry and immunohistochemistry are complementary, and that flow cytometry is superior to immunohistochemistry for detecting antigens/epitopes present in low amounts.

Aged↗

PubMed1998

The immunogenic properties of melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last 6 years significant progress has been achieved in the identification of melanoma-associated antigens recognized by cytotoxic T lymphocytes. These antigens belong the three main groups: tumor-associated testis-specific antigens (MAGE, BAGE, GAGE and PRAME), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2) and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). In this review, we have summarized the available data concerning the characterization of melanoma-associated antigens with focus on their immunogenic and protective properties. The development of a strong immune response against differentiation antigens is limited by the existence of tolerance against these 'self' antigens, permitting the involvement of only T cells with low affinity T cell receptors. Among the melanoma differentiation antigens, only gp100 has been shown to be a tumor regression antigen. The testis-specific antigens such as MAGE and PRAME should potentially be highly immunogenic antigens. They contain several potential HLA class I binding epitopes and are present only in the testes which are not accessible to the cells of the immune system due to the lack of direct contact with the immune cells and the lack of HLA class I expression on the surface of germ cells. But only 2 patients have been found who responded to these antigens in vivo, indicating their genuinely low immunogenicity. A comparison of the predicted secondary structures of these two groups of antigens (testis-specific and differentiation antigens) revealed enrichment of long alpha-helical stretches in the testis-specific antigens. We hypothesize that such highly organized structures could diminish the efficiency of the protein unfolding--a necessary step in the proteolytic cleavage by proteasomes--and, therefore, could be responsible for the low immunogenicity of these proteins. In this case, modifications decreasing the stability of these proteins might be a means to improve the immune response against these potentially therapeutically useful antigens.

Amino Acid Sequence↗

PubMed1998

Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE).

Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.

Cells, Cultured↗

PubMed1998

Immunogenicity, immunosensitivity and cell surface adhesiveness of tumour vaccines carrying an inserted CD80 gene.

Cell surface adhesiveness, immunogenicity and immunosensitivity of tumour vaccines modified by the CD80 gene transfection was examined and compared to that of the parental MC12 murine sarcoma. Insertion of the CD80 gene substantially enhanced the adhesiveness of the genetically modified tumour cells to nylon wool non-adherent (T) but not to nylon wool adherent (B) lymphocytes. The increased adhesive interaction could be inhibited by anti-CD80 monoclonal antibody. CD80+ transfectants were more sensitive to the cytotolytic effect of MC12-immune splenocytes and IL-2-activated spleen cells than the parental MC12 sarcoma. Similarly, spleen cells from syngeneic mice immunized with CD80+ transfectants displayed a higher cytolytic activity when allowed to react with MC12 cells than splenocytes from mice immunized with the parental MC12 cells. These results suggest that a positive correlation exists among the expression of the CD80 molecules, T cell adhesion to the genetically modified cells, immunosensitivity of the CD80+ transfectants and the capacity of the transfectants to activate cytolytic, tumour-reactive effector cells in vivo. This correlation provides a rationale for gene therapy based on the construction of CD80- modified tumour vaccines.

Animals↗

PubMed1997

Complete scanning of the CDK4 gene by denaturing gradient gel electrophoresis: a novel missense mutation but low overall frequency of mutations in sporadic metastatic malignant melanoma.

The cyclin-dependent kinase 4 (CDK4) is a key component in regulation of the mammalian cell cycle. The recent discovery of a common missense mutation (Arg24Cys) in both sporadic and familial forms of malignant melanoma strongly supports the candidacy of CDK4 as a proto-oncogene. To study further the role of CDK4 in melanoma pathogenesis, we have established a method based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to scan the CDK4 gene for point mutations. By analyzing the entire coding sequence of the CDK4 gene in 56 sporadic metastatic malignant melanomas, we identified a novel missense mutation, Asn41Ser. This mutation was also found in the germline of the patient who had no family history of melanoma. Analysis of a tumor-derived cell line demonstrated equal expression of the mutant and wild-type CDK4 alleles, together with lack of functional p16. Our findings suggest that an oncogenic mechanism of the CDK4-Asn41Ser variant would be different from the CDK4-Arg24Cys variant. Altogether, our data demonstrate that point mutation of CDK4 is a rare event in melanoma pathogenesis.

Asparagine↗

PubMed1997

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma.

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.

Base Sequence↗

PubMed1997

Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3.

Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3. Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-gamma receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2(+)-binding protein annexin II, and proteins from the hsp70 family. Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides. These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy.

Animals↗

PubMed1997

A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma.

Mutations in the N-, K-, and H-ras genes are key events in the process of carcinogenesis of many human cancers and may serve as important targets for therapeutic intervention. We developed a simple diagnostic method that in one step and within 5 hr determines the mutational status of any of the 3 ras genes in a given tumor sample. The method combines polymerase chain reaction (PCR) with denaturing gradient gel electrophoresis (DGGE) and allows simultaneous mutation scanning of 6 regions covering "hot-spot" codons 12, 13 and 61 of the 3 ras genes. The sensitivity of the assay was demonstrated by the analysis of control mutations, either naturally occurring or created by site-directed mutagenesis. We further demonstrate that unambiguous identification of ras mutations can be achieved by heteroduplex analysis in denaturing gradient gels, circumventing sequence analysis. We applied the method to establish the mutational status of all 3 ras genes in 123 samples of B-cell non-Hodgkin's lymphoma. Altogether, one diffuse large B-cell lymphoma and one B-cell chronic lymphocytic leukemia (B-CLL) harbored a mutation (G12S and G12A, respectively) in the K-ras gene, and one B-CLL harbored a mutation (Q61R) in the N-ras gene. We therefore conclude that ras mutations only contribute rarely, if at all, to carcinogenesis in B-cell non-Hodgkin's lymphoma.

Base Sequence↗

PubMed1997

Expression of transporter associated with antigen processing 1 and 2 (TAP1/2) in malignant melanoma cell lines.

TAP1 and TAP2 molecules are involved in the transport of peptides prior to their association with class I molecules and are mandatory for efficient antigen presentation. To investigate whether loss of expression of TAP1 or TAP2 is a likely mechanism of immune escape in malignant melanoma, TAP1 and TAP2 mRNA was analyzed by RT-PCR in 39 melanoma cell lines expressing at least 2 of the known melanoma-associated antigens, tyrosinase, Melan-A/MART-1, gp100, MAGE-1 and MAGE-3. All 39 cell lines expressed both TAP1 and TAP2 at the mRNA level. To investigate other factors potentially involved in immune escape, the expression of LMP2, LMP7, HLA class I molecules, beta2-microglobulin (beta2m) and specific HLA-A alleles was evaluated by RT-PCR and FACS analyses. All 39 cell lines expressed LMP2, LMP7 and beta2m. A single cell line (FM37) had lost the expression of class I molecules, and this same cell line showed loss of expression of the HLA-A2 heavy chain. No cell lines showed loss of expression of the HLA-A1 heavy chain. Based on our studies of in vitro established cell lines, loss of TAP1/2 or LMP2/7 expression does not appear to be a common mechanism of immune escape in malignant melanoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Single-step DGGE-based mutation scanning of the p53 gene: application to genetic diagnosis of colorectal cancer.

We present a simple nonradioactive assay based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE), which allows comprehensive mutation scanning of the entire coding sequence and splice junctions of the p53 gene in one analytical step. The key features of the present method are (1) all fragments can be amplified using one common PCR protocol; (2) all fragments can be scanned for mutations on a single "broad-range" denaturing gradient gel; and (3) all fragments were tailored by the attachment of appropriate GC clamps and otherwise manipulated to consist of only two melting domains in order to maximize resolution of mutations by DGGE. The entire procedure starting with a sample of genomic DNA can be completed within 6 hr and has the potential to detect any sequence variation, irrespective of its location in the p53 gene. The mutation detection sensitivity was demonstrated by the analysis of 26 constructed control mutations distributed over the whole of the p53 gene. We have applied the method to genetic diagnosis in 43 cases of colorectal cancer. Overall, a point mutation, microdeletion, or microinsertion was found in 26 (61%) of the tumors. In addition to missense and frameshift mutations within exons 4-8, a 20-bp insertion in exon 11 was found in one sample, illustrating the importance of comprehensive gene scanning for reliable diagnosis.

Aged↗

PubMed1997

Analysis of T cell receptor AV and BV chain gene expression by infiltrating lymphocytes in Spitz naevi and in halo naevi.

Spitz naevi and halo naevi are benign melanocytic lesions that share many histological features with malignant melanoma. All lesions are characterized by a brisk infiltration of lymphocytes, mainly of the T cell subtype, and halo naevi are known to undergo spontaneous regression. Since the benign nature of Spitz naevi and halo naevi might therefore be caused by specific T cell responses against tumour-associated antigens, it was found of interest to characterize this T cell response in detail. A reverse transcriptase-polymerase chain reaction (RT-PCR)-based method adapted for analysis of paraffin-embedded material combined with Southern blot analysis has been used to analyse the T cell receptor (TCR) AV and BV repertoires of infiltrating lymphocytes in 14 different melanocytic lesions. The results have shown that only a few particular TCRAV and TCRBV regions are expressed in each lesion. To evaluate the T cell response, it is of interest to know the HLA-type of the analysed lesions, since most melanoma-specific effector lymphocytes are CD8+ cytotoxic T cells and therefore HLA class I-restricted. As blood samples were not available from any of these patients, an RT-PCR method using HLA-A2-specific primers was used to analyse for the presence of this allele. The preferentially expressed TCRAV genes were sequenced, and this analysis showed that the high expression of these TCRAV genes was due to a clonal or oligocional expansion of T cells. In summary, the expression of relatively few TCR variable regions indicates a clonal expansion of T cells.

Adolescent↗

PubMed1997

Escape from host-antitumor immunity.

Cancer cells may express proteins recognizable by the individual's immune system as foreign because they are either tumor-specific or not expressed at high levels in normal tissues to which the host is tolerant. There is now much evidence that tumors can be immunogenic, that is, that they frequently express antigens in a form recognizable by the host immune system. This has been shown not only in experimental animals but also for spontaneously occurring human tumors. Tumors therefore may progress by evolving variants that can evade immune responses or by developing other strategies to "escape" the immune response. The purpose of this review is to consider the current status of knowledge concerning these different tumor escape strategies.

Animals↗

PubMed1996

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide.

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Carcinoma, Squamous Cell↗

PubMed1996

The p16-cyclin D/Cdk4-pRb pathway as a functional unit frequently altered in melanoma pathogenesis.

The p16Ink4/CDKN2, D-type cyclins, their partners Cdk4/Cdk6, and pRb constitute a G1 regulatory pathway commonly targeted in tumorigenesis. Genetic, immunochemical, and functional cell cycle analyses showed abnormalities of this pathway in each of 22 human melanoma cell lines examined. Normal melanocytes and all melanoma lines expressed Cdk4, Cdk6, and cyclins D1 and D3. The tumor suppressors p16Ink4/CDKN2 and pRb were lost in 17 and 4 cases, respectively, due to various genetic mechanisms, including transcriptional block of p16 and nonsense mutations of RB1. Ectopic expression of p16 prevented S-phase entry of Rb+/p16- but not Rb-deficient melanoma lines. The SK29-MEL-1 cell line harboring an R24C mutation in Cdk4 expressed wild-type pRb and overabundant p16, the latter preventing endogenous Cdk6 but not Cdk4 from associating with cyclin D1. Microinjection of cyclin D1-neutralizing antibody arrested the SK29-MEL-1 cells in G1, whereas pl6 did not, indicating that the cyclin D1/Cdk4-R24C complex is required for G1 progression, and the resistance of the complex to p16 in vivo. These data strongly support the candidacy of Cdk4 as a novel proto-oncogene, provide further evidence for the p16-cyclin D/Cdk-pRb pathway as a functional unit, and suggest that deregulation of this checkpoint may represent a common step in the multistep progression of sporadic malignant melanomas.

Base Sequence↗