Biomedical subjects

P Telleman

Publications and source records attributed to P Telleman.

13 recordsLinked to original sources

PubMed2004

One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates.

Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices.

Amines↗

PubMed2004

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays.

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray trade mark slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.

Base Sequence↗

PubMed2003

Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter.

The integration of complete analyses systems "on chip" is one of the great potentials of microfabricated devices. In this study we present a new pressure-driven microfabricated fluorescent-activated cell sorter chip with advanced functional integration. Using this sorter, fluorescent latex beads are sorted from chicken red blood cells, achieving substantial enrichments at a sample throughput of 12000 cells s(-1). As a part of the sorter chip, we have developed a monolithically integrated single step coaxial flow compound for hydrodynamic focusing of samples in flow cytometry and cell sorting. The structure is simple, and can easily be microfabricated and integrated with other microfluidic components. We have designed an integrated chamber on the chip for holding and culturing of the sorted cells. By integrating this chamber, the risk of losing cells during cell handling processes is eliminated. Furthermore, we have also developed integrated optics for cell detection. Our new design contributes to the ongoing efforts for building a fully integrated micro cell sorting and analysing system.

Animals↗

PubMed2001

Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer.

A minority of breast cancers is characterized by lymphoplasmacytic infiltrates that have been correlated with improved patient survivals. The association of improved prognosis with plasmacytic infiltrates has been classically linked with the rare medullary carcinoma subtype but is also evident in the smaller infiltrated fraction of the more abundant nonmedullary (not otherwise specified) tumors. It is our hypothesis that these plasma cell (PC) infiltrates represent a host humoral response driven by one or more tumor-derived neoantigens. As the index study group, two primary medullary carcinoma tumors were examined. Immunophenotyping confirmed a large number of IgG PCs in contradistinction to normal breast, which typically contains a lesser number of mainly IgA isotypes. IgG heavy and light chains were expressed as combinatorial phage Fab libraries. VH and VL sequences showed a preponderance of clonal groups in both patients, as identified by germ-line gene usage and junctional mutation patterns. Panning of phage Fab libraries against purified antigens excluded Her2/neu and p53 as the eliciting antigen, and failure of clonal enrichment by cell panning suggested that the neoantigen was not membrane expressed or was expressed at low levels. Cognate, original VH+VL pairs were obtained by single cell PCR of tumor PCs, which showed overlap with the pooled IgG libraries. Tumor-derived IgG V genes exhibited mutational patterns that were consistent with antigenic selection and affinity maturation. Where examined, IgG1 was the predominant isotype, consistent with a T-dependent (i.e., protein) antigen. From these data, we infer that the breast tumor PC infiltrates of the medullary carcinoma subtype are compatible with an autogenic tumor neoantigen-driven humoral immune response.

Antibodies, Neoplasm↗

PubMed2000

Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.

B-Lymphocytes↗

PubMed2000

The role of the Brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile.

The Brambell receptor (FcRB) mediates functions of both immunoglobulin G (IgG) transport, transmitting immunity from mother to young, and IgG protection, making IgG the longest surviving of all plasma proteins. Reflecting its role as transport receptor (termed FcRn, for neonatal rat intestine, the tissue from which it was first cloned), FcRB is expressed antenatally in the rabbit, mouse and rat fetal yolk sac and in human placental syncytiotrophoblasts, and neonatally in the intestinal epithelium of mice and rats. Reflecting its role as protection receptor (FcRp), FcRB is expressed in the vascular endothelium throughout life, where it protects IgG from the on-going catabolic activities of this tissue. FcRB detected in hepatocytes was hypothesized to mediate transport of IgG from serum to bile, thus potentially extending the transport expression (FcRn) of this receptor beyond the perinatal period. Our results show serum-to-bile transport of IgG to be unaffected in mice functionally deleted for FcRB. Accordingly, the hypothesis is rejected that FcRB functions as transport receptor (FcRn) in liver. The default conclusion is that FcRB in hepatocytes functions as FcRp, serving to protect IgG from catabolism in hepatocytes that accompanies the endocytic activity of these cells. We conclude that there remains to date no evidence of an FcRn-like transport function of the Brambell receptor beyond the perinatal period, after which the FcRp function of the receptor predominates, paralleling the endocytic activities of the associated tissues.

Animals↗

PubMed2000

Direct immunoprecipitation of antigen with phage displaying immunoglobulin fragment.

Phage libraries may display hormones, receptors, antibody fragments, etc., by fusion to phage envelope proteins. This report describes the direct precipitation of phage-Fab-antigen complexes by polyethylene glycol precipitation, resulting in highly selective and efficient recovery of antigen from complex mixtures without nonspecific protein contamination. The method demonstrates efficiency and specific recovery of phage-Fab-antigen complexes from a background of a complex mixture of unrelated proteins as may occur in the analysis of biological specimens. This simple, fast, and effective method allows isolation and characterization of target antigens, with no need to further process Fab or sFv, and may reasonably be extended to isolate any interacting partner molecule for any displayed protein.

Animals↗

PubMed1997

An optimized method for cell-based phage display panning.

BACKGROUND: Traditional methods of phage display panning, bind purified antigen to plates or other solid phases to which libraries are then applied, followed by vigorous washings in detergent-supplemented buffers to select for specific phage Fab. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. OBJECTIVES: To develop a model antigen system employing whole cells rather than purified protein immobilized on a substrate; to optimize methods for phage display panning using a cell-based system. RESULTS: Specificity of binding of phage Fab to antigen on cells was demonstrated by output titer and by flow cytometry. Output titers showed a plateau and binding advantage, after four washes, corresponding to removal of most non-specifically bound phage. Enrichment advantage was independent of input phage number. Longer incubation times, to cell tolerance, improved specific binding. Temperature had modes impact as a variable in the panning and washing. An increase in output titers paralleled enrichment for specific phage Fab. CONCLUSION: An optimized method applied to whole cells can productively enrich specific phage Fab in mixtures with large excesses of non-specific phage Fab over several rounds of panning.

Animals↗

PubMed1996

Spectrum of spontaneously occurring mutations in the HPRT gene of the Chinese hamster V79 cell mutant V-H4, which is homologous to Fanconi anemia group A.

The mitomycin C (MMC)-hypersensitive Chinese hamster V79 cell mutant V-H4 has a cellular phenotype similar to Fanconi anemia (FA), and has been shown to be homologous to FA group A. To examine consequences of the defect in V-H4 cells on spontaneous mutagenesis, we studied the frequency and nature of spontaneous mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus in this mutant and the parental V79 cells. The mutation rates expressed as the number of mutations per cell per generation were 8.7 x 10(-7) and 3.7 x 10(-7) for V-H4 and V79 cells respectively. The molecular spectrum of 42 spontaneous hprt mutants of V-H4 cells was determined and compared with the previously described spectrum of spontaneous mutations at the HPRT locus of Chinese hamster V79 cells. The spectra of spontaneous mutations in the hprt gene of both cell lines are predominated by base pair substitutions and splice mutations. Among the base changes, V-H4 shows a larger frequency of transitions (13/42; 31%) than transversions (3/42; 7%), whereas in V79 transversions are observed more often than transitions (P < 0.001; Wilcoxon test). The frequency of splice mutations in V-H4 (17/42; 40%), which affects exon 4 almost exclusively, is not significantly different from V79. The fraction of deletions in V-H4 is low (6/42; 14%), and comparable to the level in V79. This is in contrast with the published molecular spectrum of spontaneous hprt mutants in FA (group D) cells, which consists predominantly of deletions.

Animals↗

PubMed1995

A new complementation group of mitomycin C-hypersensitive Chinese hamster cell mutants that closely resembles the phenotype of fanconi anemia cells.

Three Mitomycin C (MMC)-hypersensitive mutants (CL-V1B, CL-V5B, and CL-V101B) were isolated from Chinese hamster V79B cells by the replica plating technique. In comparison to the parental cell line, CL-V1B, CL-V5B, and CL-V101B show about a 22-, 32-, and 13-fold increased sensitivity to MMC, respectively (judged by the D10). These mutants are also sensitive to other DNA cross-linking agents, such as 1,2,3,4-diepoxybutane (9-, 19-, and 12-fold, respectively) and cis-diamminedichloroplatinum(II) (17-, 12-, and 6-fold, respectively). CL-V5B and CL-V101B display an exclusive sensitivity to DNA cross-linking agents, whereas CL-V1B also shows an increased sensitivity to monofunctional alkylating agents, such as methyl methanesulfonate (3-fold) and ethyl methanesulfonate (2-fold), and UV254mm (2-fold). Approximately 2-3-fold higher levels of spontaneous chromosomal aberrations are found in these three mutants in comparison to wild-type V79B cells. At a MMC survival level of 80%, CL-V5B demonstrates a 16-fold higher level of MMC-induced chromosomal damage than V79B. Despite phenotypical heterogeneity within this group of mutants, hybrid clones derived after fusion remained MMC sensitive, indicating that these mutants belong to the same complementation group. To determine whether the mutants represent a new complementation group among other Chinese hamster cell mutants that also display hypersensitivity to MMC, CL-V1B cells were fused with mutants representing different complementation groups i.e., irs1, irs3, irs1SF, UV20, UV41, V-H4, and V-C8 cells. In all cases, the derived hybrids regained MMC sensitivity similar to wild-type cells, indicating that the CL-V1B mutant represents a new complementation group. The phenotype of CL-V1B, CL-V5B, and CL-V101B cells closely resembles the phenotype of Fanconi anemia cells, suggesting that these hamster mutants could be defective in a gene that is involved in this disorder.

Alkylating Agents↗

PubMed1994

Mutagenic response and repair of cis-DDP-induced DNA cross-links in the Chinese hamster V79 cell mutant V-H4 which is homologous to Fanconi anemia (group A).

Previously, it has been shown that the V-H4 mutant of Chinese hamster V79 cells is homologous to Fanconi anemia (FA) group A cells. This hamster cell mutant shows a specific sensitivity to DNA cross-linking agents; therefore, the induction and repair of DNA cross-links were studied in V-H4 and wild-type V79 cells after cis-DDP treatment by the DNA alkaline elution technique. A significant difference in repair of these lesions in V-H4 and wild-type cells was observed. After the cis-DDP treatment (24 h) about 3 times more cross-links remained in V-H4 cells in comparison to the parental V79 cells. These results indicate that the process of cross-link repair in V-H4 cells is hampered when compared to that of wild-type cells. To assess the effect of slower removal of DNA cross-links on the mutability of V-H4, the induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase locus (HPRT) by cis-DDP was studied in V-H4 and V79 cells. Despite the increased cytotoxicity of cis-DDP to V-H4 cells, the mutation induction at the HPRT locus was not significantly different in both cell lines, but when the frequency of the hprt mutants was plotted against survival, hypomutability was observed in V-H4 cells after the cis-DDP treatment.

Animals↗

PubMed1993

Genetic diversity of mitomycin C-hypersensitive Chinese hamster cell mutants: a new complementation group with chromosomal instability.

A Chinese hamster cell mutant (V-C8) isolated previously, which is approximately 100 fold more sensitive to mitomycin C (MMC) than its parental wild-type V79 cells (judged by D10 values), was further characterized. V-C8 cells exhibit an increased sensitivity towards other cross-linking agents, such as cis-DDP (approximately 40-fold), DEB (approximately 30-fold), and also to adriamycin (approximately 5-fold), and the monofunctional alkylating agents: MMS (approximately 5-fold) and EMS (approximately 6-fold). V-C8 cells show a higher level induction of chromosomal aberrations by cross-linking agents (MMC, cis-DDP, and DEB) and an increased level of spontaneous chromosomal aberrations in comparison to the wild-type V79 cells. To determine whether the V-C8 mutant represents a new complementation group among Chinese hamster cell mutants that also display the extreme sensitivity to MMC, V-C8 cells were fused with irs1, irs1SF, UV20, UV41, and V-H4 cells. In all cases, the derived hybrids regained the MMC sensitivity similar to wild-type cells, indicating that the V-C8 mutant belongs to a new sixth complementation group.

Animals↗

PubMed1992

Effect of ploidy on transcription levels in cultured rat aortic smooth muscle cells.

Aging and hypertension increase the number of polyploid smooth muscle cells (SMC) in a blood vessel. We assessed the effect of ploidy on the transcription of several genes in SMC cultures derived from newborn and adult rats. In diploid and tetraploid subcultures of SMC from newborn rats, RNA expression of the genes assayed is linked with ploidy. However, when phenotypically different SMC cultures derived from newborn and adult rats were compared, transcription levels varied from gene to gene and not linked with the ploidy. Thus, differences in gene expression due to polyploidy are superimposed on those due to other phenotypical features.

Actins↗