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Biomedical subjects

L Amiot

Publications and source records attributed to L Amiot.

At least 19 recordsLinked to original sources
PubMed2007

Elevated levels of soluble non-classical major histocompatibility class I molecule human leucocyte antigen (HLA)-G in the blood of HIV-infected patients with or without visceral leishmaniasis.

The non-classical class I major histocompatibility complex molecules human leucocyte antigen (HLA)-G have been shown to play a role in HIV persistence, but no data are available on the expression of the soluble forms HLA-G5 and sHLA-G1 in HIV-infected patients with and without opportunistic infections. The soluble HLA-G isoform was measured with an enzyme-linked immunosorbent assay (ELISA) method in plasma from 94 subjects: 31 HIV-1-seropositive, 17 with visceral leishmaniasis (VL), seven with both VL and HIV-1 infection and 39 healthy HIV-seronegative subjects. Between groups, the frequency of sHLA-G positivity was statistically different: 81% of HIV-infected patients were positive, as were 57% of HIV-Leishmania infantum co-infected patients, 35% of HIV-seronegative patients with VL and 3% of healthy controls. Levels of the soluble forms of the immunomodulatory molecules HLA-G are elevated during HIV infection. In HIV-Leishmania co-infected patients, sHLA-G secretion could contribute to the tolerogenic environment and to Leishmania immune evasion.

AIDS-Related Opportunistic Infections↗
PubMed2002

Lung macrophages and dendritic cells express HLA-G molecules in pulmonary diseases.

HLA-G is selectively expressed in extravillous trophoblast of human placenta, which does not express classical HLA-A and -B molecules. Several studies report the role of HLA-G as a molecule involved in immune tolerance. By interacting with NK and T cells inhibitory receptors, HLA-G may downregulate their cytotoxicity functions. To appreciate the biologic and clinical relevance of HLA-G expression in lung diseases, HLA class I and HLA-G expression were analyzed in a panel of 36 ex vivo neoplastic tissues and 8 non-neoplastic lung tissues. Immunohistochemical analysis was performed using a pan-HLA class I antibody (W6/32) and three different specific anti-HLA-G antibodies (87G, MEMG/9 and 4H84). These findings demonstrated that HLA-G products were not expressed in pulmonary structural cells. However, HLA-G molecules were detected in activated macrophages and dendritic cells infiltrating lung carcinomas (33%) and nontumoral pulmonary diseases (25%). HLA-G expression was not correlated with classical HLA alterations. No statistical correlation was found between HLA-G expression and clinical or biologic parameters except high tumor size. The expression of HLA-G in myelo-monocytic cells infiltrating lung pathologic tissues could alter antigenic presentation and contribute to decrease immune response efficiency, subsequently favoring the progression of tumoral or inflammatory processes.

Dendritic Cells↗
PubMed2002

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood.

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

Antigens, CD34↗
PubMed2001

Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC.

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.

Antigen-Presenting Cells↗
PubMed2000

Modulation of HLA-G antigens expression in myelomonocytic cells.

As trophoblast cells and macrophages share cellular characteristics, we investigated the expression of HLA-G antigens during the myelomonocytic differentiation. Analyses with the 87G and 16G1 monoclonal antibodies demonstrated that HLA-G was not expressed in peripheral blood monocytes, in in vitro differentiated dendritic cells and macrophages, and in resident mononuclear phagocytes infiltrating healthy tissues. Conversely, activated macrophages and dendritic cells localized in tumoral biopsies of some lung carcinomas expressed HLA-G antigens. Induction of HLA-G expression at the cell surface of the monohistiocytic cell line U 937 with different cytokines strongly suggests that cytokines secreted during inflammation may be involved in this specific upregulation. Bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also expressed HLA-G molecules. In vitro, we thus demonstrated that HLA-G antigens are produced during viral reactivation in the macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. Our data suggest that inflammatory processes in lung tissues, like tumoral transformation and HCMV acute infection, are likely to induce HLA-G molecules in infiltrating macrophages and dendritic cells. The expression of molecules capable of downregulating both the innate and adoptive immunity could be a mechanism that helps tumoral and HCMV infected cells to escape immune response.

Acute Disease↗
PubMed1999

Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells.

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.

Cells, Cultured↗
PubMed1999

HLA-DR inhibits granulocytic differentiation without inducing apoptosis of CD34 cells.

Hematopoietic progenitors express HLA-DR molecules. However the significance of HLA-class II molecules on CD34+ cells remains unknown. The primary role of HLA-class-II molecules is antigen presentation although a second role, that of signal transduction, has been established in B cells. The role of HLA-DR in hematopoiesis was examined by determining the ability of CD34+ progenitor cells to differentiate to "Colony Forming Unit Granulocyte-Macrophage" (CFU-GM) and "Burst Forming Unit Erythrocyte" (BFU-E) in the presence of anti-HLA-DR monoclonal antibody. We observed a reduction in the number of CFU-GM which was due in part to down regulation of granulocyte rather than monocyte differentiation. These observations suggest that HLA-DR signals can regulate myelopoiesis. We point out especially the role of the HLA-DR molecule in the switch of CFU-GM between granulocyte or monocyte lineages. Although HLA-DR mediated apoptosis has been described in mature B lymphocytes apoptosis of CD34+ cells was excluded as a mechanism.

Antibodies, Monoclonal↗
PubMed1999

HLA-G protein expression is not induced during malignant transformation.

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.

Cell Movement↗
PubMed1999

Reproducible scoring of CFU-GM and BFU-E grown in collagen-based semisolid medium after a short (3 h) training.

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

Analysis of Variance↗
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

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

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↗