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Biomedical subjects

R Fauchet

Publications and source records attributed to R Fauchet.

At least 37 records · Page 2Linked to original sources
PubMed1999

Use of the anionic dye carboxy-2',7'-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity.

Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug efflux pumps conferring multidrug resistance to tumor cells and sharing numerous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly MRP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 cells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells through inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells, although to a lesser extent, which may likely be linked to the low, but detectable, expression of MRP in these cells. Comparison of CDF retention with that of calcein demonstrated that the former dye was the most efficiently effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P-glycoprotein was not found to alter cellular CDF labelling whereas it strongly impaired calcein staining. These results indicate that CDF is a substrate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

HLA-G class I gene expression in normal and malignant hematopoietic cells.

The class Ib HLA-G gene encodes for a molecule which is selectively expressed in fetal placental cells. Fetomaternal tolerance could be partially explained by the interactions between HLA-G molecules and KIR receptors of decidual NK cells. To determine whether the presence of HLA-G antigens might constitute a factor of immune tolerance during the tumoral process, we compared the expression of the HLA-G gene in normal and malignant hematopoietic cells. Despite a HLA-G transcriptional activity in several lymphocytes and monocytes, no antigens are found at the cell surface or in the cytosol using the specific HLA-G mAb, 87G. This lack of expression does not appear modified in malignant hematopoietic cells. However, treatment of the monohistiocytic cell line U937 with different cytokines enabled the expression of HLA-G antigens to be induced. We suggest that the potential induction of HLA-G molecules in monocytic malignant cells following secretion of cytokines may constitute a factor of immune tolerance in patients.

Cell Line, Transformed↗
PubMed1998

Multidrug resistance analysis in lymphoproliferative disease of large granular lymphocytes.

Multi-drug resistance (MDR) phenotype contributes to the ineffectiveness of chemotherapy. P-glycoprotein (PgP) and lung resistance protein (LRP) are proteins implicated in chemoresistance. We analysed the expression of PgP and LRP respectively in 17 and 15 cases of lymphoproliferative disease of granular lymphocytes (LDGL) including 10 cases of clonal large granular lymphocytic (LGL) leukaemia, six cases of oligoclonal (n = 5) and polyclonal (n = 1) CD3+ lymphoproliferation and one case of CD3- NK lymphocytosis. Functional PgP activity, as determined by Rh123 dye efflux assay, was found in all the patients. The mean percentage of effluxing cells was 47 +/- 22%, compared to 35 +/- 8% on normal lymphocytes (P<0.04). The efflux was blocked in the presence of verapamil, a PgP revertant agent. A high proportion of CD57+ cells (66 +/- 10%) from these patients expelled Rh123. Functional PgP activity was associated with expression of MDR1 mRNA. By using immunocytochemistry, LRP expression was detected in 11/15 patients (73%). 7/10 LGL leukaemia patients presented a LRP+/Efflux+ phenotype and 5/7 had LRP+/Efflux+/MDR1 mRNA+ phenotype. These findings suggest that the PgP+/LRP+ phenotype is frequently observed in LDGL. Its clinical relevance in aggressive cases remains to be determined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Loss of HLA molecules in B lymphomas is associated with an aggressive clinical course.

Major histocompatibility complex class I molecule expression is reduced in some malignant tumours permitting escape from immune surveillance and is therefore associated with a poor prognosis. Seven cases of non-Hodgkin lymphomas out of 300 cases of malignant lymphoproliferative disorders totally lacked expression of class I molecules as determined by flow cytometry. Clinical data confirmed a particular aggressiveness of these cases with frequent extra-nodal involvement, a poor international prognostic index, a histological high grade and a poor outcome leading to early death in five of the seven cases. A previous diagnosis of follicular lymphoma characterized by bcl-2 rearrangements was made in four of these cases. HLA-G (class Ib gene), which is reported to bind killer inhibitory receptors on NK cells, was absent from the cell surface. However, it was detected in three out of four cases at the mRNA level with transcripts encoding soluble forms. Additional analysis revealed other abnormalities: class II was negative in four out of the seven NHL cases and decreased expression of beta2 microglobulin was observed in all cases. Peptide transporter proteins (TAP1) were detected in various degrees by immunocytochemistry. These observations showed that total lack of class I or class II molecules is a rare event in NHL and is associated with a poor prognosis. This could support a role for specific autologous T cells in immune surveillance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Value of early blood Th-1 cytokine determination in predicting severity of acute pancreatitis.

BACKGROUND: Early evaluation of the severity of acute pancreatitis (AP) requires measurement of many variables within 48 h after admission. Septic complications (SC) are frequent, and preliminary studies have highlighted the value of prophylactic antibiotherapy; however, single and reliable predictive markers of sepsis are not yet available. The aim of this study was to assess the value of determining early blood Th-1 cytokines and their natural antagonists (interleukin-6 (IL-6), IL-1, IL-1ra, and the soluble form of tumor necrosis factor (sTNF) receptors RI and RII) to predict the severity and SC during AP. METHODS: Thirty-seven patients with AP were prospectively included; 25 of them had severe AP, including 8 with SC. Serum cytokines were measured 48 h and 72 h after the onset of AP with an enzyme-linked immunosorbent assay. The optimal severity or SC diagnostic thresholds was determined using receiver operative curves. RESULTS: Severe AP in accordance with the Atlanta criteria were better predicted by C-reactive protein and IL-6 serum determination, albeit these levels could not predict absolutely the death of two patients. In severe AP cases (n = 25) the IL-1 to IL-1-ra ratio was lower in cases further complicated by sepsis ((6+/-4) 10(-3) versus (34+/-13) 10(-3), P < 0.05); moreover, sTNF RI (2497+/-270 pg/ml versus 2133+/-611 pg/ml, P < 0.05) and RII (3751+/-400 pg/ml versus 3045+/-509 pg/ml, P < 0.05) were higher in AP characterized by further SC. The IL-1 to IL-1-ra ratio and IL-1 concentration were dramatically decreased within the first 48 h ((0.4+/-0.4) 10(-3) versus (30+/-11) 10(-3), P < 0.05, and 0.3+/-0.3 versus 15+/-3 ng/l, P < 0.05) in patients with further infection of the pancreatic necrosis (n = 3). The SC diagnosis was better anticipated by an IL-1 to IL-1-ra ratio lower than 5 x 10(-3) or by an sTNF RI higher than 1750 pg/ml and sTNF RII higher than 2750 pg/ml, and the infection of the pancreatic necrosis by an IL-1 concentration <2 ng/l or an IL-1 to IL-1-ra ratio <2 x 10(-3). CONCLUSION: Besides severity markers, IL-1, IL-1-ra, and sTNF RI and RII should be considered in base-line AP assays and, if confirmed by larger studies, could help to screen patients at risk for SC and candidates for prophylactic antibiotherapy with a good negative predictive value.

Acute Disease↗
PubMed1998

Multidrug resistance in aggressive lymphoproliferative disorders of T and natural-killer origin.

The measurement of rhodamine 123 (Rho123) efflux in hematological malignancies, using flow-cytometry, provides an accurate assessment of multidrug resistance (MDR) of both P-glycoprotein and MRP. While their normal counterparts display high levels of PgP and Rho123 efflux, we investigated the MDR status of marked T/NK proliferations. When diagnosed according to natural killer (NK) markers (CD16, CD56, CD57) 8 of nine NK lymphoproliferative disorders (LPD) were markedly positive (3 NK non Hodgkin's lymphomas (NHL), 1 NK lymphoproliferative disease of large granular lymphocytes (LGL), and 5 T/NK LGL). These results are in accordance with the observed response to chemotherapy in the treated cases. Mature T LPD (prolymphocytic leukemia (PLL), and NHL) cells gave varying results, as did cells from Sezary syndromes. Marked Rho123 efflux was detected in the two cases of T-PLL suggesting the expression of MRP as previously described. Immature T-lymphomas or leukemias (6 cases) were all negative. These data should be considered in relation to NK proliferations which clearly display an MDR phenotype and therefore raise the question, of the relevance of this phenotype in normal cells, and secondly of the negativity of immature T-LPD. The latter could indicate that MDR inhibitors may be superfluous in the initial treatment of acute lymphoblastic leukemia (ALL). Finally the resistance to treatment of T-ALL or mature T cells LPD invokes the importance of exploring other mechanisms of drug resistance such as the lung resistance related protein (LRP).

Antigens, CD↗
PubMed1998

Differential expression and activity of P-glycoprotein and multidrug resistance-associated protein in CD34-positive KG1a leukemic cells.

CD34+ acute myeloid leukemias generally respond poorly to chemotherapy when compared to CD34- myeloid leukemias. In order to contribute to the analysis of the mechanisms involved in this drug resistance, expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), two drug efflux pumps conferring multidrug resistance, have been investigated in the CD34+ KG1a leukemic myeloid cell line and in two CD34- K562 and HL60 leukemic myeloid cell lines. Reverse transcription-polymerase chain reaction and dye efflux assays revealed that KG1a cells express P-gp but not MRP whereas neither P-gp nor MRP were detected in K562 and HL60 cells. In addition, KG1a cells were demonstrated to display resistance to anticancer drug substrates for P-gp such as vincristine and daunorubicin and to poorly accumulate vincristine. These results indicated that P-gp, in contrast to MRP, is expressed and functional in the drug-resistant CD34+ KG1a cell line, that may constitute a useful cellular model to analyze the constitutive chemoresistance of CD34+ acute myeloid leukemias.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Spontaneous recovery from severe parvovirus B19 pure red cell aplasia, in a heart transplant recipient, as demonstrated by marrow culture.

A 61-year-old heart transplant recipient with parvovirus B19 infection, presented as a severe pure red cell aplasia (PRCA) with hemoglobin level of 5 g/dl. Both blood and bone marrow cells were positive for parvovirus B19 DNA, whereas specific immunoglobulins IgG and IgM were not informative. Bone marrow smears revealed erythroid hypoplasia without giant pronormoblasts. Autologous and allogenic bone-marrow cultures revealed a high inhibition by patient's serum on BFU-E growth whereas the number of CFU-GM were normal. Spontaneous remission of the anemia was observed despite the persistence of severe immunodeficiency as demonstrated by development of a monoclonal EBV lymphoproliferative disorder two months later. The "recovery" serum reversed the initial serum BFU-E inhibiting property. This case pinpointed the usefulness of blood or marrow cultures in parvovirus B19 infection of immunocompromised patients without normal Ig responses, as in other PRCA. Further, it argues that the usual immunoglobulin therapy may not be necessary in order to obtain a viral clearance.

Bone Marrow Cells↗
PubMed1997

CD3- CD56+ non-Hodgkin's lymphomas with an aggressive behavior related to multidrug resistance.

CD56 expression has been reported previously in some non-Hodgkin's lymphoma (NHL) characterization. They principally involve the nasopharynx, are related to Epstein-Barr virus (EBV), and may be classified as either T- or non-T-natural killer (NK) cells according to CD3/T-cell receptor (TCR) status at the genomic or protein level. The present study reports three cases of non-nasal NK-NHL with the following characteristics: an agressive clinical behavior, heterogenous morphological data evoking pleomorphic T-cell malignant lymphoma, a non-T-NK phenotype using flow cytometry, and immunochemistry. The three cases were CD56+ without membrane expression of specific T markers (CD3, CD5, and TCR). Heterogenous results were observed concerning different antigens: CD2, CD4, CD8, CD16, CD94, CD122, TiA1, perforin, and granzyme B. There was no evidence of detectable clonal TCR gene rearrangement with polymerase chain reaction. No NK activity was detected in the two tested cases, and no relation was found with EBV. Multidrug resistance investigations suggest that agressive clinical findings could be related to MDR1 gene expression as confirmed by MDR1 mRNA detection, MDR1 gene product (Pgp) expression, and a functional multidrug resistance study using rhodamine efflux by flow-cytometry.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Inapparent polycythemia vera: an unrecognized diagnosis.

PURPOSE: The Polycythemia Vera Study Group (PVSG) has established useful criteria for the diagnosis of polycythemia vera. In some circumstances, an increase of plasma volume (PV) masks that of red cell mass (RCM), with hemoglobin (Hb) and hematocrit (Ht) remaining normal. This defines the concept of inapparent polycythemia. PATIENTS AND METHODS: One hundred and three patients seen in the hematology unit with the diagnosis of polycythemia vera were studied. There were 55 males and 48 females with a median age of 59 years. Ninety-five patients fulfilled the PVSG criteria. Spontaneous erythroid colonies and low serum erythropoietin level confirmed the diagnosis in the 8 other cases. Patients were classified according to Hb and Ht level. RESULTS: Group A consisted of 85 patients with increased Hb and Ht defined, respectively, by Hb > 18 g/dl, Ht > 0.52 in males and Hb > 16 g/dL, Ht>0.47 in females. Group B included 18 patients (17%) with inapparent polycythemia vera (IPV) defined by a normal Hb and Ht value at diagnosis. In this group, the reasons to perform RCM were as follows: splenomegaly associated with increased platelets and/or leucocytes counts (n = 8), portal vein thrombosis (n = 5), increased platelets or leucocytes counts without splenomegaly (n = 3), and isolated splenomegaly (n = 2). The two groups were balanced in terms of age, sex, leucocyte, serum iron, and platelet level. Hemoglobin and Ht levels were significantly different between the two groups. The difference between the PV was indeed highly significant. The mean PV increase was + 9.5% (nL < +20%) in group A versus + 36.3% in group B (P < 0.00005). Red cell mass was not different between the two groups. CONCLUSIONS: Increased Hb or Ht should constitute the sole criteria for RCM determination. In the context of portal vein thrombosis, isolated hyperleucocytosis, thrombocytosis, or splenomegaly, a RCM should be performed. The frequency of IPV remains to be specified but the diagnosis of polycythemia vera is probably underestimated.

Adult↗
PubMed1997

Leiden factor V mutation in four patients with small bowel infarctions.

The Leiden factor V mutation is observed in 20% of unexplained lower limb venous thromboses and involves substitution of the arginine residue at position 506 by glutamine (R506Q). It is known to decrease the anticoagulant activity of activated protein C. This case report describes 4 cases of small bowel infarction (SBI) associated with the presence of this mutation. Two cases of arterial and 2 cases of venous SBI were observed. Extensive assessment excluded the usual causes of SBI and plasma hypercoagulation syndrome (antithrombin III, protein C, and protein S deficiency and myeloproliferative syndrome). An abnormal resistance to activated protein C was observed. Molecular analysis consisting of polymerase chain reaction amplification and digestion with MnlI showed that 2 patients were heterozygous and 2 were homozygous for the R506Q mutation. Despite familial history of thrombosis in only 1 patient, first- and second-degree relatives of 2 patients also had the presence of the mutation. Examination for the presence of abnormal resistance to activated protein C should be part of the etiological assessment of SBI. Its presence may warrant consideration of long-term anticoagulant therapy, especially for patients with shortened small bowel who are treated by home parenteral nutrition with deep venous access.

Adult↗
PubMed1997

Rat liver biliary epithelial cells support long-term production of haemopoietic progenitors from human CD34 cells.

In this study we report the supportive activity of rat liver epithelial cells (RLEC) on human haemopoiesis in the absence of exogeneously supplied growth factors. RLEC is a rat cell line derived from primitive biliary cells with epithelial characteristics which induce the long-term differentiation of hepatocytes through cell-cell contacts. We have established the ability of these cells to sustain long-term survival and multilineage differentiation of human haemopoietic progenitors from unfractionated bone marrow and growth-factor mobilized peripheral blood cells, and from human CD34+ and CD34+ CD38- haemopoietic cells, with a higher efficiency than the murine MS-5 stromal cell line: the numbers of committed progenitors recovered from RLEC cocultures after 8 weeks were 3-fold higher than from MS-5 cocultures, with an unusually high BFU-E production. Furthermore, using diffusible insert cultures, we demonstrated that, despite the lack of strong adhesive interaction between haemopoietic cells and RLEC, physical proximity was absolutely required for optimum stimulation of LTC-IC by RLEC. Taken together, these results show that biliary epithelial cells support human haemopoiesis and cause speculation that common mechanisms might be used by RLEC to regulate both the hepatocyte and the haemopoietic progenitors differentiation.

ADP-ribosyl Cyclase↗
PubMed1997

CpG methylation patterns in the 5' part of the nonclassical HLA-G gene in peripheral blood CD34+ cells and CD2+ lymphocytes.

A dominant goal of research focused on the nonclassical human leukocyte antigen G (HLA-G) gene is to understand the molecular mechanism involved in its limited expression. In the present report, we examined DNA methylation as a potential regulatory mechanism of HLA-G transcription in two cell types of the adult lymphomyeloid lineage: CD2+ lymphocytes express several mRNA isoforms while transcripts are undetectable in CD34+ hematopoietic cells. The methylation status of 63 CpG sites in the promoter and in the 5' CpG island was established using bisulfite-treated genomic DNA sequencing. Methylation was first analyzed by the direct sequencing of bisulfite-treated and amplified products. The general patterns of CpG methylation in the 5' part of the gene were found to be similar for CD34+ cells and CD2+ lymphocytes: the distribution of methylation was not uniform across the 63 CpG sites. In the promoter region, both CpG dinucleotides were partially or fully methylated whereas in the CpG island, several CpG sites were totally demethylated. Unexpectedly, in HLA-G positive CD2+ lymphocytes, a great number of CpG dinucleotides displayed a higher frequency of methylation relative to that found in CD34+ cells. However, the sequence analysis of cloned products revealed that the molecules have different methylation patterns which suggests that the HLA-G gene is differentially expressed in CD2+ cells. Our results suggest that methylation is not the sole mechanism that achieves the repression of HLA-G transcription in immature CD34+ cells.

Adult↗
PubMed1996

Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells.

In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5' CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of beta2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.

Alternative Splicing↗