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M Platzer

Publications and source records attributed to M Platzer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Genomic Organization of the ATM gene.

The ATM gene was recently identified and found to be responsible for the genetic disorder ataxiatelgiectasia. The major ATM transcript is 13 kb. Using long-distance PCR, we determined the genomic structure of this gene and identified all of its exon-intron boundaries. The ATM gene spans approximately 150 kb of genomic DNA and consists of 66 exons. The initiation codon falls within exon 4. The last exon is 3.8 kb and contains the stop codon and a 3'-untranslated region of about 3600 nucleotides.

Ataxia Telangiectasia↗
PubMed1996

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP).

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.

Cell Line↗
PubMed1995

Up-regulation of monocytic IL-10 by tumor necrosis factor-alpha and cAMP elevating drugs.

It is well established that endotoxin [lipopolysacharide (LPS)] induces pro-inflammatory cytokine production in monocytes, which is followed by secretion of the anti-inflammatory cytokine, IL-10. IL-10 down-regulates inflammatory response [tumor necrosis factor (TNF)-alpha, IL-1, IL-6, IL-8] as well as IL-10 synthesis itself. We wondered whether pro-inflammatory cytokines such as TNF-alpha may be involved in the regulation of human IL-10 synthesis. TNF-alpha induced de novo IL-10 mRNA expression in a dose-dependent manner but no IL-10 protein in human peripheral blood mononuclear cells. Furthermore, LPS-induced IL-10 gene and protein expression was significantly inhibited by neutralizing anti-TNF-alpha mAb. On the basis of these results, we conclude that TNF-alpha is involved in the up-regulation of its antagonist IL-10. Paradoxically, drugs that effectively inhibit expression of TNF-alpha via the elevation of intracellular cAMP level (iloprost, pentoxifylline, prostaglandin E2 and N6,2-O-dibutyryl cAMP) augmented the endotoxin-induced IL-10 synthesis at both protein and mRNA levels. In order to provide a basis for the analysis of the transcriptional regulation of the human IL-10 gene, we isolated a fragment of the human IL-10 gene containing 1308 bp of the 5' non-coding sequence. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation, including a cAMP responsive element which could explain the cAMP-mediated effects. The lack of a NF-kappa B-like binding site in the human sequence suggests a NF-kappa B-independent mechanism of TNF-alpha-induced IL-10 gene activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Differential promoter activity in benign and malignant human cells of skin origin.

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.

Carcinoma↗
PubMed1994

Extension of incomplete cDNAs (ESTs) by biotin/streptavidin-mediated walking using the polymerase chain reaction.

In the last 2 years thousands of new partial cDNAs or expressed sequence tags (ESTs) have been identified by single pass sequencing methods. It is expected that this number will further increase in order to help to isolate all human genes. However, the scientific value of partial cDNA fragments is limited unless they are used as tools for isolating and sequencing their full length parent molecules. Conventional library screening methods are tedious and not very effective in achieving this goal. We present a modified PCR technique which allows rapid isolation of the ends of partial cDNA fragments in vitro using a biotin/streptavidin capture procedure. Our method has several advantages over the RACE technique, is very specific, and allows to frequently sequence the final product directly without subcloning. We also show that cDNA walks can be obtained from partial sequences as short as 26 bp.

Bacterial Proteins↗
PubMed1994

5' noncoding sequence of human IL-10 gene obtained by oligo-cassette PCR walking.

A fragment of the human IL-10 gene starting from a Hind III site and including 744 previously unknown bases of the 5' regulatory region was isolated by oligo-cassette PCR walking and the nucleotide sequence was determined. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation.

Animals↗
PubMed1990

Transfection by DNA-nuclear protein HMG1 complexes: raising of efficiency and role of DNA topology.

We have developed a novel and efficient transfection method based on the introduction of foreign DNA into mammalian cells in form of complexes of vector DNA with the nuclear protein HMG1. In this study, it is shown that a stabilization of the complexes against dilution dissociation by addition of soluble CaCl2 or by excessive HMG1 enhances the transfection efficiency. Furthermore, there are no differences in the transfection abilities between the 3 topological DNA forms, viz., supercoiled, open relaxed and linear DNA, if delivered to cells as HMG1-DNA complexes. It is further shown that transfection-inactive complexes of the core histones with foreign DNA can be activated in transfection by the addition of HMG1.

Animals↗
PubMed1989

The influence of antithyroid drugs and iodine on thyroid cell MHC class II antigen expression.

We have investigated the influence of antithyroid drugs (methimazole and propylthiouracil) and sodium iodide on the expression of major histocompatibility (MHC) class II antigen expression in human and rat thyroid cells. While methimazole and propylthiouracil significantly inhibited lectin-induced MHC class II (HLA-DR) antigen in crude human thyroid monolayer preparations these drugs had no influence on gamma-interferon induction of class II antigens in similar cell preparations. Hence, antithyroid drugs probably had a direct effect on thyroid monolayer T cells which are known to effect the lectin induction of MHC class II antigen via T-cell secretion of the cytokine, gamma-interferon (IF). Sodium iodide, similarly, had no direct influence on human thyroid cell MHC class II antigen expression induced by gamma-IF. However, iodide significantly inhibited the gamma-IF-induced expression of MHC class II antigens in a proliferating rat thyroid cell clone derived from FRTL-5 cells (clone 1B-6). In the rat thyroid cell clone this antagonistic action appeared to be exerted via inhibition of TSH-induced proliferation. These data add further support to the multifactorial nature of the profound immunosuppressive influence of antithyroid drugs in autoimmune thyroid disease and experimental thyroiditis. In contrast, iodides may have a major, and often overlooked, influence on thyroid cell proliferation and antigen expression.

Animals↗
PubMed1989

Reovirus induction of MHC class II antigen in rat thyroid cells.

We have previously demonstrated that cultured rat thyroid cells do not exhibit constitutive expression of major histocompatibility (MHC) class II antigens. Using reovirus types 1 and 3, we infected 1B-6 cells (a cloned derivative of the Fisher rat cell line FRTL-5), and found a dose-dependent induction of thyroid cell MHC class II antigen expression as determined by laser flow cytometry and FITC-labelled OX-6 anti-RT1.B. As the number of viral particles/cell used for the infections increased from 5 to 100, the number of antigen positive cells increased, in reovirus type 3 infections to 50%, and in reovirus type 1 infections to 15%. Kinetic studies indicated that MHC class II antigen expression continues to increase three days after infection. Viral infection and the resulting MHC class II antigen expression may allow presentation of thyroid antigen to the immune system and participate in the initiation of autoimmune thyroid disease.

Animals↗
PubMed1988

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes.

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.

Animals↗
PubMed1988

Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection.

The aim of this study was the search for a method of vector packaging using natural chromatin constituents. The interaction of the chromosomal non-histone protein HMG1 with a vector plasmid (pLTEneo) was studied by sedimentation analysis and electron microscopy at physiological salt concentration. At high protein input the complexes exist in a condensed, monodisperse form sedimenting with 80 S irrespective of the supercoiled or relaxed conformation of DNA. Saturation binding is already observed at much lower input ratios. Dilution of 80 S complexes results in decondensation of the complexes. In the decondensed complex form, HMG1 binds in a bead-like manner to specific DNA regions. Condensation by HMG1 is sufficient to introduce the vector into mammalian cells without the need for unphysiological additives. The transfection rates were similar to or even higher than those obtained by the calcium phosphate coprecipitation technique.

Animals↗
PubMed1988

Retention of cyclic AMP response to TSH in a cloned human thyrocyte/T cell hybridoma (HY2-15).

Our observation of a human T cell leukemia cell (Molt 4) demonstrating low affinity thyroid-stimulating hormone (TSH) responses, as evidenced by generation of cyclic AMP, led us to test Molt 4 cells as a suitable partner for immortalizing high affinity TSH receptors present on human thyroid cells. Therefore, we generated a hybridoma (HY2-15) by a fusion between thyroid monolayer cells from a patient with Graves' disease, and a hypoxanthine-aminopterin-thymidine (HAT)-sensitive variant of this human T cell leukemia line, Molt 4-8AGR. The hybrid nature of HY2-15 was confirmed by DNA histograms using propidium iodide and flow cytometry. Karyotyping showed the HY2-15 cells to have five sets of chromosomes and human leukocyte antigen (HLA) class I determination revealed the presence of an additional HLA class I antigen (A2) not present on the Molt 4 partner cells. The established, cloned, hybridoma cells showed a greater than 30-fold increase in cyclic AMP release after stimulation with bovine TSH (bTSH, 1 mU/ml) with a minimum detectable stimulating dose of less than 10 microU/ml bTSH. However, no other thyroid-specific functions could be detected. Furthermore, HY2-15 cells failed to express HLA class II antigens either constitutively or in response to recombinant human gamma interferon (IF) and a variety of other stimuli, data similar to the Molt 4 partner cells but in contrast to human thyroid cells which show high sensitivity to gamma IF. The preservation of highly sensitive TSH responsiveness in a proliferating cell offers a unique approach to the study of human TSH receptor function.

Cell Division↗
PubMed1988

HLA-DR gene expression in a proliferating human thyroid cell clone (12S).

We have used a retroviral vector carrying the adenovirus E1A oncogene and the neomycin phosphotransferase gene to establish a human thyroid-derived cell line that exhibits TSH-mediated cAMP generation as well as the differential expression of HLA class II antigens in response to recombinant gamma-interferon. Twenty-two-week gestation, histologically confirmed, human fetal thyroid was collagenase digested, cultured as a monolayer, and infected directly with 12S or 13S E1A-containing retrovirus constructs. Infected clones (n = 30) were selected in a hormone-supplemented medium containing bovine TSH (bTSH; 1 mU/ml), 10% fetal bovine serum, and 0.5 mg/ml G418 antibiotic. A rapidly growing clone (designated 12S) was chosen for detailed analysis over 18 months of continuous culture. The 12S clone was sensitive to less than 10 microU/ml bTSH when assessed by extracellular accumulation of cAMP, but TSH had no influence on 72-h incorporation of [3H]thymidine. Clone 12S responded to recombinant human gamma-interferon (1-10(4) U/ml) by induction of HLA DR alpha-chain-specific mRNA and the surface expression of HLA-DR antigen detected by fluorescein isothiocyanate-labeled monoclonal antibody to nonpolymorphic HLA-DR regions using flow cytometry. These studies indicate the potential for immortalizing human thyroid cells for use as targets of anti-TSH receptor immune responses and for long term studies of human throcyte HLA gene regulation.

Adult↗
PubMed1987

Cloning the Fisher rat thyroid cell line (FRTL-5): variability in clonal growth and 3,'5'-cyclic adenosine monophosphate response to thyrotropin.

To investigate the stability of FRTL-5 cells we cloned stock cells by limiting dilution in a 6-hormone medium with bovine TSH (bTSH) (10 mU/ml) (6H medium). One third of the wells with irradiated fibroblast feeder cells developed FRTL-5 colonies. One hundred and twenty clones were obtained, of which 29 (8%) developed sufficiently for functional analysis. Clones were tested for growth responses to bTSH stimulation as evidenced by 72-h [3H]thymidine uptake and TSH receptor activation by cAMP generation. Clonal growth responses to bTSH were of three types: dose-related growth increase, absence of growth stimulation, and stimulation by bTSH in concentrations up to 100 microU/ml and inhibition of growth above 100 microU/ml. All clones tested showed evidence of extracellular cAMP accumulation in response to bTSH. However, sensitivity to bTSH varied from 1 to 100 microU/ml and maximum cAMP secretion with 1 mU/ml bTSH varied from 2 to 13 pmol/ml. Whereas certain clones showed high sensitivity to bTSH with respect to both growth and cAMP responses (e.g. 1B-6), there were clones which showed disparity in this relationship, as evidenced by poor growth dependency but high cAMP responses or by growth stimulation yet insensitivity with respect to cAMP secretion. These data demonstrate that the FRTL-5 line contains cells with variable responsiveness to bTSH. Whereas the FRTL-5 line is heterogeneous and subject to developmental variation, cloning by limiting dilution allows the derivation of highly bTSH-sensitive cells, such as 1B-6, from the stock cultures.

Animals↗
PubMed1987

Induction of rat thyroid cell MHC class II antigen by thyrotropin and gamma-interferon.

Major histocompatibility (MHC) class II antigen expression by thyroid epithelial cells has been widely implicated in the pathogenesis of autoimmune thyroid disease. We have examined rat MHC (RT1) class II antigen gene regulation in 1B-6 cells, cloned in our laboratory from the Fisher rat thyroid line FRT-L5. 1B-6 cells are TSH dependent for growth and proliferation, and are responsive to more than 5 microU/ml bovine TSH (bTSH) in terms of extracellular cAMP accumulation. Recombinant rat gamma interferon (gamma IF; 1-1000 U/ml) treatment for 5 days together with bTSH (10(3) microU/ml) was able to induce class II antigen expression in up to 90% of 1B-6 cells, as detected by a murine monoclonal antibody to rat MHC class II antigen (FITC-OX-6). Total cellular RNA was examined in Northern blot analyses using an HLA-DR alpha-chain-specific RNA probe, which shares 78% sequence homology with the rat RT1.D alpha-chain gene. A 1.4-kilobase mRNA transcript was detected in gamma IF-stimulated cells, but not in untreated cells. Dose-response studies of MHC class II gene expression, using a cytoplasmic RNA slot blot technique for alpha-chain mRNA levels and FITC-OX 6 monoclonal antibody for detection of MHC class II antigen expression, indicated 1B-6 sensitivity to gamma IF in the range of 10-100 U/ml (in the presence of 10(3) microU/ml bTSH) and to bTSH in the range of 10-100 microU/ml (in the presence of 10(2) U/ml gamma IF). These data demonstrate dual control of MHC class II antigen gene expression by gamma IF and bTSH in a differentiated rat thyroid cell. Clone 1B-6 represents a powerful means of analyzing reproducibly and in long term studies the regulation of thyroid cell MHC class II gene expression.

Animals↗
PubMed1987

Lymphokine regulation of HLA-DR gene expression in human thyroid cell monolayers.

Studies were conducted to examine the regulation of HLA class II gene expression in human thyroid cells in vitro. Normal human thyroid cells cultured in the absence of lectin or gamma-interferon stimulation lacked detectable HLA-DR cell surface antigen, although low levels of DR alpha-chain-specific mRNA were present. Cyclosporine A, known to inhibit lymphokine production, inhibited basal as well as lectin-mediated increases in levels of DR alpha-chain-specific mRNA and DR surface antigen expression on normal human thyrocytes. Cyclosporine had no effect on the induction of DR antigen gene expression by recombinant gamma-interferon. These data suggested that lectin enhancement of DR antigen expression in human thyroid cells may be mediated by a lymphokine(s) produced in primary human thyroid cell monolayers. This suggestion was confirmed by studies that demonstrated the abrogation of lectin responsiveness by antibody directed against gamma-interferon. Indirect immunofluorescence studies using flow cytometric analyses identified 1.6 +/- 0.2% (mean +/- SD) of cells in primary thyroid cultures as T lymphocytes, a potential source of lymphokine production. Cells derived from thyroid follicular adenomas and carcinomas demonstrated reduced lectin-mediated increases in DR antigen expression compared to normal thyroid cells. DR expression could be enhanced in these lectin-treated cells, however, by T cell coculture. Dose-response studies demonstrated that human thyroid cells were as sensitive to gamma-interferon induction of DR antigen expression as human monocyte/macrophages. These results indicate that human thyroid cell HLA-DR antigen gene expression is sensitive to low levels of lymphokines, such as gamma-interferon; an intrathyroidal T cell population, which may serve as a source of lymphokine(s), remains associated with thyroid epithelial cells in primary thyroid cultures; and lymphokine-thyroid cell interactions may be implicated in the immunopathology of human autoimmune thyroid disease.

Antigens, Neoplasm↗
PubMed1986

hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells.

Our previous studies have indicated specificity cross-over between LH/hCG and the TSH receptor. We have now analyzed the ability of the TSH-dependent Fisher Rat thyroid cell line (FRTL-5) to proliferate in a differentiated state under the influence of highly purified hCG (hCG-CR121). TSH receptor activation and growth induction were observed after 7 days suspension from a TSH-induced growth phase. The effect of 10 ug hCG-CR121 was equivalent to 500 +/- 43 (mean +/- SEM) uIU of human TSH (2nd IRP, 80/558) with reference to growth stimulation, as judged by 72-h [3H]-thymidine uptake and to approximately 15 +/- 35 uIU human TSH with reference to receptor activation as judged by cyclic AMP accumulation. These data demonstrate specificity cross-over by hCG for the TSH-dependent FRTL-5 cell line and suggest that hCG has greater growth-stimulating than thyroid-stimulating potential when compared with human TSH.

Animals↗