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Biomedical subjects

P Paul

Publications and source records attributed to P Paul.

At least 37 records · Page 2Linked to original sources
PubMed2000

Modulation of HLA-G expression in human thymic and amniotic epithelial cells.

Expression of the nonclassical HLA class I antigen, HLA-G, is tightly regulated. HLA-G physiologic expression is mostly restricted to some placental and thymic cell types. Only few established cell lines express HLA-G in vitro. Cytokine-induced expression of HLA-G is hardly observed and also depends on the cell lineage. We assessed expression and cytokine regulation of HLA-G in primary cultures derived from human thymus and amnion epithelial cells, which also express HLA-G in vivo. We show that HLA-G cell surface expression is maintained, but decreases gradually, in primary cultures derived from human thymus and amnion epithelial cells. We also show that IFN-gamma re-induces HLA-G cell surface expression and upregulates classical class I gene expression in both primary cultures and in a thymus derived cell line. We further show that IFN-gamma also upregulates levels of HLA-G transcripts in TEC primary cultures. This study provides evidence that IFN-gamma induction of HLA-G expression occurs in the human amnion and the thymus, and is mediated at the transcriptional level in these tissues. These results also suggest a role for the microenvironment in regulating HLA-G in vivo gene expression in the thymus and amnion membrane.

Amnion↗
PubMed2000

Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells.

The nonclassical HLA-G primary transcript is alternatively spliced to generate several mRNAs that have the capacity to encode four membrane bound isoforms, namely HLA-G1, -G2, -G3, and -G4 and two soluble isoforms HLA-G5 and -G6. We aimed at defining the capacity of full length and truncated soluble HLA-G transcripts to be translated in human cell lines. Our study of HLA-G alternative transcripts in various human tissues led us to identify a new splice variant of the HLA-G mRNA, named G7, in which open reading frame continues in intron 2. Due to the presence of a stop codon within intron 2, HLA-G7 transcripts retain the capacity to be translated as soluble truncated HLA-G proteins bearing the alpha1 domain linked to two specific aminoacids encoded by intron 2. Expression vectors containing cDNAs encoding HLA-G5, -G6, and -G7 isoforms were transfected into human cell lines. The presence of translated HLA-G5, -G6, and -G7 proteins was detected in protein extracts of transfected cells by Western blot and immunoprecipitation, but only the full length HLA-G5 soluble isoform could be clearly detected as a secreted protein in both transfected cells supernatants and body fluids.

Alternative Splicing↗
PubMed2000

The X1 box of HLA-G promoter is a target site for RFX and Sp1 factors.

HLA-G gene regulation was investigated with regards to homologies among the pathways regulating both classical MHC class I and MHC class II gene expression. They include four conserved cis-acting regulatory elements located in the proximal promoter region referred to as the W/S/Z box, the X box that is comprised of the X1 and X2 halves, and the Y box with an inverted CCAAT site. The X1 box is the binding site for the ubiquitous RFX complex consisting of three subunits; the X2 box is bound by the X2BP/ATF/CREB family factors. The basic S-X-Y regulatory module interacts with CIITA, which is expressed constitutively in APCs, but may be inducible in others cell types by IFN-gamma. Within HLA-G gene promoter the only conserved motifs are S and X1 boxes. We thus investigated the binding capacity of the HLA-G X box in comparison to that of HLA-DRA and HLA-E. We demonstrate that X2 box mutations in HLA-G promoter affect the binding of ATF/CREB family factors and may privilege the X2 box to access by other shared factors. The X1 box is the target for RFX complex and an additional factor we identified as Sp1. We propose that the X region in the HLA-G gene promoter might participate to the combination of factors which play a role in HLA-G gene activation.

Cell Extracts↗
PubMed2000

Use of pattern standard deviation instead of corrected pattern standard deviation in Anderson's criteria.

PURPOSE: To determine whether the use of pattern standard deviation instead of corrected pattern standard deviation, as part of Anderson's criteria, makes a difference in categorizing a single field printout using the full threshold strategy on the Humphrey's Field Analyzer. MATERIALS AND METHODS: Forty-eight patients with glaucomatous field defects of varying severity underwent full threshold perimetry on the Humphrey's Field Analyzer. The third field of one eye was used for the study. The agreement between CPSD and PSD alone was determined. Each field was then categorized using Anderson's criteria first using CPSD and then PSD. The kappa statistic was used to determine agreement in both situations. RESULTS: The agreement between CPSD and PSD alone, as determined by kappa, was 0.77. The kappa statistic for categorization using Anderson's criteria was 0.82; the weighted kappa was 0.92. CONCLUSION: There was almost perfect agreement using the PSD instead of CPSD as part of Anderson's criteria. This substitution seems valid for the spectrum of field defects used in this series, at least for the full threshold programs.

Adult↗
PubMed2000

Heat shock and arsenite induce expression of the nonclassical class I histocompatibility HLA-G gene in tumor cell lines.

The nonclassical histocompatibility class I gene HLA-G has a tissue-restricted expression. To explore mechanisms involved in HLA-G transcriptional regulation, we have investigated the effect of stress, including heat shock and arsenite treatment, on HLA-G expression in tumor cell lines. We show that stress induces an increase of the level of the different HLA-G alternative transcripts without affecting other MHC class I HLA-A, -B, -E, and -F transcripts. A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) on stress conditions was further identified within the HLA-G promoter. Considering the ability of HLA-G to modulate the function of immunocompetent cells, we hypothesize a new feature of HLA-G as a signal regulating the immune response to stress.

Arsenites↗
PubMed2000

A comparison of concurrent and retrospective verbal protocol analysis.

In verbal protocol analysis, verbalization can occur either during decision making (concurrent data) or after (retrospective data). Although both methods have advantages and disadvantages, no empirical research has focused on a direct comparison. This study compared the effectiveness of concurrent and retrospective data for revealing the human decision making process. In general, the concurrent protocol analysis method outperformed the retrospective method. Not only was the number of concurrent protocol segments elicited higher than that of retrospective protocol segments, but concurrent data provided more insights into the decision-making steps occurring between stimulus introduction and the final choice outcome. However, retrospective protocols offer an interesting advantage: More statements about the final choice are provided in retrospective protocols than in concurrent protocols.

Data Collection↗
PubMed1999

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study.

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor d-threo-1-phenyl-2- decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by >80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Amino Acid Sequence↗
PubMed1999

Heterogeneity of HLA-G gene transcription and protein expression in malignant melanoma biopsies.

Nonclassical MHC class I HLA-G antigen expression is tissue specific and is thought to play a role in tolerance of the semiallogeneic fetus by the maternal immune system. Ectopic expression of HLA-G by tumor cells provides them with an additional mechanism of escape from immunosurveillance by host cytotoxic effector mechanisms. The aim of this study was to assess the expression of nonclassical HLA-G antigens in ex vivo human melanoma biopsies. HLA-G mRNA levels corresponding to both membrane-bound and soluble protein isoforms were analyzed in tumor specimens obtained from primary or metastatic melanomas of 23 patients. High levels of HLA-G transcription were detected in tumor specimens in 5 of 23 patients and found to be comparable in both lymph node and skin metastases. HLA-G mRNA transcript levels at tumor sites in 18 of these patients were compared with those in samples of their own healthy skin and were higher in the tumor tissue in 12 patients. Differential expression of mRNA transcripts corresponding to soluble and membrane-bound HLA-G was also observed in some tumor biopsies. HLA-G protein expression was detected in tumors that exhibited high levels of HLA-G transcription by immunofluorescence of frozen sections and Western blot analysis of both tumor and healthy skin biopsies, using anti-HLA-G-specific monoclonal antibodies. This work provides evidence that HLA-G gene transcription and protein expression can be up-regulated ex vivo in melanoma. Our finding that several of the tumors studied expressed high levels of HLA-G provides additional clues as to how a tumor can be selected in vivo to escape from cytotoxic antitumor responses, constituting a new parameter to be considered in the design of therapeutic approaches aimed at enhancing antitumor immune responses.

Adult↗
PubMed1999

Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase.

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an approximately 38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an approximately 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of approximately 10,000-fold purified Golgi GCS with the same reagents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.

Amino Acid Sequence↗
PubMed1999

The immunotolerance role of HLA-G.

HLA-G is a non-classical MHC class I molecule involved in immune tolerance. We present our results concerning the effects of HLA-G on the cellular immune response, where it impairs both NK and T cell functions. We describe the NK inhibitory properties of HLA-G ex vivo, demonstrating its role in materno-fetal tolerance, which is supported by our in vitro studies using membrane-bound HLA-G1- and HLA-G2-transfected cells and a full-length soluble HLA-G molecule. We also report how HLA-G interacts at the T cell level, here exemplified by its inhibitory effect on both T cell allogeneic proliferative and Ag-specific CTL responses.

Cell Division↗
PubMed1999

HLA-G expression: immune privilege for tumour cells?

The non-classical class I antigen HLA-G has been shown to play a role in foeto-maternal tolerance. It interacts with inhibitory receptors to down-regulate natural killer and T cell cytotoxic functions. We here report our investigations on HLA-G expression in melanoma cells and its implication in the induction of immune tolerance to tumours. We provide the first evidence that both malignant human melanoma cell lines and ex vivo biopsies can exhibit high levels of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns. We further demonstrated that melanoma cells that express HLA-G inhibit NK cytolysis. We thus propose that HLA-G expression may impede the elimination of malignant cells by anti-tumour immune effector cells, constituting a newly described mechanism by which tumour cells may evade immunosurveillance.

Disease Models, Animal↗
PubMed1999

Role of HLA-G versus HLA-E on NK function: HLA-G is able to inhibit NK cytolysis by itself.

Recent studies have shown that endogenous HLA-E molecules are stabilized on the cell surface upon the expression of HLA-G which contains within its leader sequence, a nonapeptide capable of binding with the HLA-E/beta2m complex. Since HLA-E was found to be the major ligand for the CD94/NKG2A inhibitory receptor, we determined the role of HLA-G versus HLA-E on NK lysis inhibition. We showed that K562 cells transfected with HLA-G1 cDNA are protected from NK lysis by direct interaction between HLA-G1 and killing inhibitory receptor(s). This NK lysis inhibition is not dependent on HLA-E expression, since no HLA-E protein was detected on K562 cells; HLA-G1 is therefore able to inhibit NK lysis by itself.

Cytotoxicity, Immunologic↗
PubMed1999

Molecular mechanisms controlling constitutive and IFN-gamma-inducible HLA-G expression in various cell types.

HLA-G molecule is thought to play a major role in down-regulating the maternal immune response by inhibiting NK and T cell cytolytic activities. We examined the molecular regulatory mechanisms that may control the restricted expression pattern of the HLA-G gene. We first analyzed protein interactions between nuclear extracts from the HLA-G-positive JEG-3 choriocarcinoma and the HLA-G-negative NK-like YT2C2 cell lines to a 244 bp regulatory element located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. This allowed characterization of cell-specific DNA-protein interactions that could account for differential cell-specific expression of the HLA-G gene. In particular two DNA-protein complexes were exclusively observed in YT2C2, suggesting that this HLA-G regulatory element is a target for putative cell-specific repressor factors. We further mapped nuclear factor binding sites to a 70 bp fragment in the upstream region of the regulatory element. We then investigated the effect of IFN-gamma on HLA-G gene expression. HLA-G cell surface expression was enhanced by IFN-gamma treatment in JEG-3 and U937 cell lines and peripheral blood monocytes while no effect was observed in tera-2 teratocarcinoma cell line. HLA-G transcriptional activity was increased only in JEG-3 and U937 cell lines. Activity of the 1.4-kb HLA-G promoter region was unchanged after IFN-gamma treatment in JEG-3 and Tera-2. These results suggest that both post-transcriptional and transcriptional mechanisms implicating IFN-responsive regulatory sequences outside the 1.4 kb-region are involved in IFN-gamma gene activation of the HLA-G gene.

Gene Expression↗
PubMed1999

HLA-G expression in human melanoma cells: protection from NK cytolysis.

Expression of the non-classical HLA-G class I antigen is physiologically restricted to a limited number of tissues including trophoblasts, and is thought to play a role in establishing tolerance of the fetus by the maternal immune system. We investigated whether ectopic expression of HLA-G could also be detected in tumor cells and confer them the ability to escape immune cytotoxic responses. High levels of all alternatively spliced HLA-G transcripts could be detected in melanoma cells by RT-PCR. Analysis of biopsies from a melanoma patient revealed a higher HLA-G transcription level in skin metastasis as compared to healthy skin, while specific amplification of the HLA-G5 transcript was only observable in the tumor. HLA-G protein expression could also be detected in two melanoma cell lines. HLA-G-positive tumors inhibit cytotoxic lysis by the NK cell line YT2C2-PR. This inhibition is not observed with B-EBV cell lines bearing matched class I specificities, and is thought to occur through interaction of HLA-G with inhibitory receptors that are distinct from known KIRs interacting with HLA-E or classical class I molecules. Together, these results confirm that HLA-G expression at the surface of tumor cells can participate in the evasion of antitumoral immune responses and favor tumor progression.

Cytotoxicity, Immunologic↗
PubMed1999

Downregulation of HLA class I gene transcription in choriocarcinoma cells is controlled by the proximal promoter element and can be reversed by CIITA.

The expression pattern of MHC class I genes in trophoblast cells at the feto-maternal interface is thought to be the basis of the maintenance of pregnancy by protecting the fetus from maternal immune rejection. Transcription of classical HLA class I genes is low or undetectable in most trophoblast cells as well as in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells. The aim of this study was to characterize the regulatory mechanisms that repress HLA-A transcription in these cell types. We show that the -335 to ATG region of the HLA-A11 gene promoter is inactive in JEG-3 and BeWo cells while able to control efficient reporter gene transcription in other cell types, indicating that this region is the target for downregulation of HLA-A expression in choriocarcinoma cell lines. The regulatory sequence involved in HLA-A gene repression was further mapped to a proximal regulatory element within the -107 to +2 ATG region of the promoter. We show that the HLA-A promoter activity cannot be induced by interferon-gamma (IFN-gamma) and that exogenous MHC class II transactivator CIITA is able to induce HLA class I promoter activity in these cells. Stringent transcriptional regulatory mechanisms, implicating the lack of basal and IFN-gamma-inducible class I promoter activity, are thus involved in the downregulation of HLA-A expression in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells.

Cell Line↗