Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells.

Multidrug resistance (MDR) in tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of surface proteins, such as P-glycoprotein (Pgp), which act as ATP-dependent molecular pumps. In a previous study, voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on resistant human osteosarcoma cells. The effects of voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the plant extract. In the present study, a panel of monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an epitope of the drug transporter in its functional conformation, was detected in voacamine-treated U-2 OS-R cells. Conversely, the expression of the epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the plant extract reacts with Pgp producing conformational changes with consequent epitope modulation. Taken together, our observations seem to demonstrate that voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated drug export, acting as a competitive antagonist of cytotoxic agents.

P-glycoprotein expression affects the intracellular concentration and antiviral activity of the protease inhibitor saquinavir in a T cell line.

A number of ATP-binding cassette proteins, which function as cellular efflux pumps, are known to be expressed on the membranes of human cells, including CD4-positive lymphocytes. It has also been shown recently that most anti-HIV protease inhibitors (PIs) are first-rate substrates of one of these membrane transporters, P-glycoprotein (Pgp). These findings raise the question of whether Pgp expression could influence HIV replication and/or affect the action of PIs. To gain new insight into this, initially unexpected, phenomenon, a study was undertaken with the aims of investigating whether treatment with saquinavir (SQV) induces Pgp expression in primary or transformed human T cell lines and, primarily, establishing whether Pgp expression could modify both the uptake of SQV and its antiviral action. Pgp expression, mainly measured by reverse transcription-PCR, was found to be variably detectable in healthy individuals, and short or prolonged SQV treatment was unable to induce or increase the expression of Pgp in a lymphoblastoid cell line or in primary lymphocytes derived from these individuals. However, further experiments, performed in a cell line with high Pgp expression (CEM(VBL100) cells) and its parental cell line (CEM cells), demonstrated that over-expression of Pgp reduces the uptake of SQV This result is consistent with the finding that CEM(VBL100) cells are less sensitive to the antiviral activity of SQV, the ID50 value (100 microM) being significantly higher than the corresponding value in parental CEM cells (4 microM).

Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells.

BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) is expressed in HIV-1 target cells, in a range of pharmacological barriers and in AIDS-associated tumours. P-gp substrates include HIV-1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug-resistant (MDR) cells. OBJECTIVES: The aim of this study was to investigate the effect on human CD4 T-lymphoblastoid CEMrev cells of saquinavir and other PIs in terms of P-gp expression and to characterize the functional and biochemical patterns of PI-induced P-gp molecules. METHODS: CEMrev cells no longer expressing detectable amounts of P-gp were cultured for a prolonged period in the presence of 10 microg/mL saquinavir (CEMsaq10) and tested for P-gp expression and function. Subsequently, CEMsaq10 cells were transferred into medium containing 15 microg/mL saquinavir (CEMsaq15) and cultured for several months. These cell lines were continuously monitored for P-gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P-gp expression in CEMrev cells. RESULTS: Compared with the drug-diluent control, the exposure of CEMrev cells to 10 microg/mL saquinavir induced, in a consistent fraction of cells (45-50%), de novo expression of functioning P-gp molecules. The transfer of CEMsaq10 cells to 15 microg/mL saquinavir was associated with a dramatic increase in P-gp expression and function (85-90% of CEMsaq15 cells expressed P-gp and effluxed P-gp dye substrates). These saquinavir-induced P-gp molecules included 75-kDa molecules as well as the classical 170-kDa form of P-gp, suggesting induction of a particular isoform of P-gp termed mini-P-glycoprotein. Conversely, ritonavir and indinavir induced transient P-gp expression in a small percentage of the CEMrev cells. CONCLUSIONS: Treatment of human CD4 T-lymphoblastoid CEMrev cells with saquinavir caused over-expression of functioning P-gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P-gp were observed in CEMsaq10 and CEMsaq15 cells during prolonged in vitro culturing, even in drug-free conditions.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

CD8(+) T-cell autoreactivity to an HLA-B27-restricted self-epitope correlates with ankylosing spondylitis.

HLA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705(+) and B*2709(+) subjects possess LMP2 236-244-specific, HLA-B27-restricted T cells, whereas only the B*2705(+) individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-gamma ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408-specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative "arthritogenic" peptide.

LC determination of indinavir in biological matrices with electrochemical detection.

A high performance liquid chromatographic (HPLC) method with electrochemical detection for the quantification of Indinavir in cell culture is described. The sample pre-treatment involved a protein precipitation procedure using acetonitrile. Chromatography was carried out on a base-deactivated reversed-phase column with an isocratic mobile phase. The method was validated with regard to specificity, linearity, limits of detection and quantitation, precision and accuracy, recovery and ruggedness. The proposed HPLC assay was utilised to directly evaluate the capability of P-glycoprotein expressing multidrug resistant cells in mediating the transport and efflux of protease inhibitor (PI) Indinavir, a basic compound in AIDS care.

Interferon-gamma up-regulates expression and activity of P-glycoprotein in human peripheral blood monocyte-derived macrophages.

P-glycoprotein (Pgp), the product of the multidrug resistance (MDR1) gene, is expressed in a variety of normal tissues but very little is known about its expression and function in cells of the immune system. In this study, we investigated the effect of interferon-gamma (IFN-gamma) on the expression and activity of Pgp in human peripheral blood monocyte-derived macrophages (MDM). We report that IFN-gamma up-regulated Pgp expression in a dose- and time-dependent manner. We show that IFN-gamma slightly increased the accumulation of MDR1 mRNA and induced a polarized redistribution of Pgp, as well as of some cytoskeletal proteins (ie, ezrin, actin, and alpha-actinin) on cell pseudopodia. Notably, confocal microscopy studies showed that Pgp and ezrin colocalized in these cellular structures. The IFN-gamma-induced Pgp up-modulation was a specific response of primary macrophages, as IFN-gamma treatment of primary lymphocytes and monocytic cell lines did not result in any increase of Pgp expression. Finally, IFN-gamma stimulated the Pgp transport activity in MDM, as rhodamine 123-efflux increased in treated cells as compared with control cultures. These results indicate that Pgp expression and activity can be up-regulated in human MDM in response to IFN-gamma. We suggest that IFN-gamma may be involved in the induction of multidrug resistance in macrophages.

The naturally occurring polymorphism Asp116-->His116, differentiating the ankylosing spondylitis-associated HLA-B*2705 from the non-associated HLA-B*2709 subtype, influences peptide-specific CD8 T cell recognition.

HLA-B27 molecules are interesting because of their strong association with ankylosing spondylitis (AS) and reactive arthritis (ReA). A pathogenetic role for these molecules has been postulated in presenting a putative "arthritogenic" peptide to CD8 T cells. The HLA-B*2709 subtype, although differing by a single amino acid (His116-->Asp116) from the widespread and strongly AS-associated subtype HLA-B*2705, is not found in patients. Since residue 116 interacts with the C terminus of the peptide, it is possible that the two subtypes differ in their antigen-presenting features. We show here that CD8 T cells can distinguish the two HLA-B27 subtypes when presenting a same epitope derived from Epstein-Barr virus-latent membrane protein 2. Moreover, alanine scanning mutagenesis analysis revealed that the peptide residues relevant for such recognition are different depending on whether HLA-B*2705 or -B*2709 molecules present the epitope. These results give support to the belief that functional differences determined by subtype-specific polymorphisms can have a pathogenetic relevance and open up a new scenario where subtle modifications within the peptide/HLA ligand might be responsible for the differential association between HLA-B27 subtypes and spondyloarthropathies.

Human natural killer cytotoxic activity is not affected by in vitro exposure to 50-Hz sinusoidal magnetic fields.

Epidemiological studies have suggested, but not demonstrated, a role of exposure to 50/60-Hz magnetic fields in increasing cancer risk in man (workers and the general population). A possible target of magnetic fields is the immune system. In particular, it is known that an important defence against cancer is represented by natural killer (NK) cells capable of killing cancer cell targets. To test this hypothesis, human NK cells, stimulated or not with phytohaemagglutinin or interleukin 2, were exposed to 50-Hz sinusoidal magnetic fields before or during the cytotoxicity test, and then mixed with a variety of target cancer cell lines (Daudi, Raji, U937, H14, IGROV, SW626, K562, HL60). The experiments were performed in two laboratories (Rome and Modena) by means of two different exposure systems. The results of both laboratories suggest that 50-Hz sinusoidal magnetic fields with flux densities up to 10 mT do not affect the cytotoxic activity of human NK cells.

[HIV seropositive pregnant women from black Africa seen at the Guy de Lorier maternity unit of Tenon hospital. Report of 33 cases]. / Les femmes enceintes séropositives (VIH+) d'Afrique noire à la maternité Guy le Lorier de l'hôpital Tenon. A propos de 33 dossiers.

There were 5503 deliveries between 1989 and 1991 in the Guy de Lorier Maternity Unit (Pr Salat-Baroux) of Tenon Hospital, Paris. These included 81 women testing HIV+ in the department with 33 asymptomatic black African women among 781 deliveries. Africans account for 14% of women delivered in the department but for 40% of seropositives in the unit, with a predilection for women from Zaïre (central Africa), accounting for 17 seropositives. They only represent 50% of all cases of African HIV+ and 20% of the unit, while they account for only 6% of the black African community and 0.8% of the maternity unit. Women from west Africa accounted for 45.5% of African seropositive cases, with Ivory Coast in first place with 24.3% while only 9% of women from Mali, accounting for 40% of African patients and 5% the units, tested positive (3 cases out of 309 patients). The mean age of seropositive patients was 23 +/- 4, pregnancies proceeded normally and there were 4 therapeutic abortions. There were 9 births by cesarean section, with no evidence of neonatal contamination, the same applying in the other 20 vaginal deliveries. Routine testing (informed consent) for HIV in these high-risk (endemic zone, drug addiction) or unrecognised seropositive patients is important whenever the opportunity presents itself (prenuptial examinations, prenatal visits, family planning, preoperative assessment) in order to attempt to lower infection rates and ensure the best possible care for mother and child when there is a wish to continue the pregnancy, but also to protect hospital staff from the risks to which they may be exposed.

Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans.

During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.

The over-expression of P-glycoprotein in K-562 and DAUDI cells, is associated with a high susceptibility to NK and LAK cells.

We evaluated the susceptibility to natural killer (NK) or lymphokine activated killer (LAK) cell-mediated cytolysis of two pairs of drug sensitive/resistant tumor cell lines which were extensively characterized at phenotypic and genotypic level. In the DAUDI cell system, the acquired capability of tumor cell variants to grow in the presence of a relatively high concentration of vinblastine (VBL) is associated with a marked increase to NK and LAK susceptibility. In contrast in the K-562 cell system, no correlation between drug-resistance, P-glycoprotein expression and susceptibility to NK or LAK activity seems to occur.

Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants.

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.

Enhancement of cell-mediated cytotoxicity by Clostridium difficile toxin A: an in vitro study.

Cells from the immune system exhibiting cytotoxic activity are able to kill tumor or infected cells in a major histocompatibility complex-restricted (cytotoxic lymphocytes) or non-restricted (natural killer cells) manner. In order to exert such a cytotoxicity they have to bind the target cell and release cytotoxic factors able to induce target cell death. Treatment of human peripheral blood mononuclear cells with toxin A from Clostridium difficile induced an enhancement of the cytotoxic efficiency of these effector cells. Morphological analysis of effector/target cell pairs seems to suggest that this could be related to an increased ability of cytotoxic effectors to establish close and intertwined contacts with target cells. These contacts involve adhesion molecules and lead to the formation of a "closed chamber" which probably improves the efficacy of lytic factors and results in an increased cytotoxicity.

Susceptibility of multidrug-resistant human T-lymphoblastoid CEM cell line to cell-mediated cytolysis.

The using of multidrug-resistant (MDR) cell variants represents one of the major obstacles to an effective cancer therapy based on the administration of cytotoxic compounds. In the present article we describe experimental procedures able to eradicate, in vitro, by using specific immunological reagent, MDR tumor cells. In an allogeneic cell system, natural killer (NK) and lymphokine activated killer (LAK) cells result effective against MDR variants of the human T-lymphoblastoid CEM cell line. Surprisingly effector cells discriminate in their lytic capacity target cells possessing a MDR phenotype. A direct relationship between the degree of relative resistance shown by target cells and cytotoxic level exerted by peripheral lymphocytes stimulated and non by IL-2 was observed. The preincubation of MDR cell variants with a monoclonal antibody (MoAb57) specific for an extramembranal epitope of P-glycoprotein induced, in presence of effector cells, a strong antibody-dependent cell-mediated cytolysis (ADCC). This phenomenon was observed only in MDR variants over-expressing in concomitance with drug-resistance high level of P-glycoprotein. In identical experimental conditions, drug-sensitive parental cells does not show valuable ADCC.