Biomedical subjects

N Gualde

Publications and source records attributed to N Gualde.

At least 19 recordsLinked to original sources

PubMed2005

The impact of eicosanoids on the crosstalk between innate and adaptive immunity: the key roles of dendritic cells.

The innate immune response is essentially the first line of defense against an invading pathogen. Through specialized receptors, known as pattern recognition receptors, especially Toll-like receptors, specialized cells of myeloid origin, including macrophages and dendritic cells (DCs) are able to phagocytose microorganisms and induce an innate inflammatory response. Although B and T lymphocytes recognize tissue antigens with high specificity, they are unable to initiate immune responses. The decision to activate an appropriate immune response is made by unique DC, the most professional antigen-presenting cells (APCs) which control the responses of several types of lymphocytes and play central role in the transition between innate and adaptive immunity. Increased secretion of inflammatory endogenous mediators such as cytokines and arachidonic acid-derived lipid mediators, also termed eicosanoids, can activate APC, particularly DC, which in turn induce an adaptive immune response. There is an increasing evidence that eicosanoids play an important role in connecting innate and adaptive immunity by acting on cells of both systems. Prostanoids, a major class of eicosanoids, have a great impact on inflammatory and immune responses. PGE(2) is one of the best known and most well-characterized prostanoids in terms of immunomodulation. Although cytokines are known as key regulators of immunity, eicosanoids, including PGE(2), PGD(2), LTB(4), and LTC(4), may also affect cells of immune system by modulating cytokine release, cell differentiation, survival, migration, antigen presentation, and apoptosis. By acting on various aspects of immune and inflammatory reactions, these lipid mediators emerge as key regulators of the crosstalk between innate and adaptive immunity.

Animals↗

PubMed2001

Granulocyte-macrophage colony-stimulating factor alone or with dacarbazine in metastatic melanoma: a randomized phase II trial.

The potential antitumoral effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) led us to evaluate GM-CSF alone or with dacarbazine (DTIC) in metastatic melanoma in first line randomized phase II. Treatment was arm A: GM-CSF: 5 microg kg(-1), bid, 14 consecutive days every 21 days and arm B: GM-CSF: 5 microg kg(-1), bid, day 2 to day 19 every 21 days and DTIC: 800 mg m(-2), day 1 of each cycle. 32 patients (pts) were included, 15 pts in arm A and 17 in arm B. All pts had visceral metastatic sites. 9 had only one metastatic site. The median number of cycles given was 2 in arm A and 3 in arm B. 100% and 89.4% of the planned dose of GM-CSF was given in arm A and arm B respectively. No objective response was obtained. 19 pts experienced at least WHO grade 3 toxicity. All pts had fever, 29 had a decrease in performance status and 23 had pain. Grade 3 toxicity were fever (38.7%), decrease in performance status (32.3%), pain (19.4%) and dyspnoea (12.5%). In this study, GM-CSF alone or in association with DTIC did not induce any antitumoral activity with subsequent toxicity.

Adult↗

PubMed2001

Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells.

Given that preliminary work has indicated that prostaglandins can play a role in modulating dendritic cell (DC) functions, we addressed the prostaglandin E(2) (PGE(2)) biosynthetic capacity of mouse DC produced in vitro from bone marrow cells. We observed production of significant amounts of PGE(2), which was reduced by at least 80% when cells were incubated in the presence of indomethacin, a COX-1 preferential inhibitor. Indeed, when tested by Western blot analysis with specific COX-1 and COX-2 antibodies, only COX-1 expression could be detected in the bone marrow (BM)-DC. For lipopolysaccharide (LPS)-treated BM-DC, inhibition of PGE(2) production by indomethacin or by NS-398 (a COX-2-selective inhibitor) used alone was less potent. After LPS treatment of BM-DC, COX-1 and COX-2 expression was potent, and inhibition of PGE(2) synthesis needed the presence of both indomethacin and NS-398. We also observed that exogenous PGE(2) diminished the expression of MHC class II molecules by BM-DC and that prostaglandin and indomethacin had antagonistic effects on cell proliferation during the mixed lymphocyte reaction using BM-DC as stimulatory cells. This assessment of PGE(2) suggests that endogenous PGE(2) produced by DC might play a role as an immunomodulating factor during the immune response. This hypothesis is sustained by the fact that IL-12 production by BM-DC is modulated by exogenous PGE(2) as well as endogenous prostaglandin, since either the addition of exogenous PGE(2) or the presence of LPS (which increases endogenous PGE(2) synthesis) decreases IL-12 production, while NS-398 (which decreases LPS-induced PGE(2) synthesis) increases IL-12 synthesis.

Adjuvants, Immunologic↗

PubMed2001

Association between immune activation and early depressive symptoms in cancer patients treated with interleukin-2-based therapy.

The relationship between immune activation and the development of early depressive symptoms were studied in 33 cancer patients undergoing cytokine therapy. Patients were treated either with subcutaneous IL-2 administered alone (n=13) or in association with IFN-alpha (n=5), or with IFN-alpha alone administered subcutaneously at low doses (n=5) or intravenously at high doses (n=10). The intensity of depressive symptoms was assessed during a clinical interview carried out before the start of cytokine therapy and five days later using the Montgomery and Asberg Depression Rating Scale (MADRS). On the same days, blood samples were collected for each patient to measure serum concentrations of cytokines (IL-6, IL-10, IL-1ra) and cytokine-receptors (sIL-2R, LIF-R). Results showed that patients treated with IL-2 or IL-2+IFN-alpha displayed concomitant mood symptoms and increased serum cytokine levels during treatment. In these patients, the intensity of depressive symptoms at endpoint was positively correlated with the increases measured in serum levels of IL-10 between baseline and endpoint. IL-10 is an anti-inflammatory cytokine that is produced in response to the production of pro-inflammatory cytokines, and thereby reflects an inflammatory response. These results support the hypothesis of close relationship between depressive symptoms and the activation of the cytokine network.

Affect↗

PubMed2001

Lowered serum dipeptidyl peptidase IV activity is associated with depressive symptoms and cytokine production in cancer patients receiving interleukin-2-based immunotherapy.

There is some evidence that treatment with interleukin-2 (IL-2) and interferon-alpha (IFNalpha) frequently induces depressive symptoms and activation of the inflammatory response system (IRS). There is evidence that major depression is accompanied by lowered serum activity of dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5), a membrane-bound serine protease which catalyses the cleavage of some cytokines and neuro-active peptides and which modulates T cell activation and the production of cytokines, such as IL-2. This study was carried out to examine the effects of immunochemotherapy with IL-2 and IFNalpha, alone and together, in cancer patients on serum DPP IV activity in relation to changes in depressive symptoms and the IRS. The Montgomery and Asberg Rating Scale (MADRS), serum DPP IV activity, and the serum IL-6, and IL-2 receptor (IL-2R) concentrations were measured in 26 patients with metastatic cancers before and three and five days after treatment with IL-2 and IFNalpha, alone or together. Treatment with IL-2 with or without IFNalpha significantly suppressed serum DPP IV activity. The MADRS scores were significantly elevated by treatment with IL-2 with or without IFNalpha, but not IFNalpha alone. The immunochemotherapy-induced decreases in serum DPP IV were significantly and inversely correlated with the increases in the MADRS. Treatment with IL-2 alone or combined with IFNalpha also elevated serum IL-6 and IL-2R. There were significant and inverse correlations between the immuchemotherapy-induced decreases in serum DPP IV and the elevations in serum IL-6 or IL-2R. In conclusion, treatment with IL-2/IFNalpha decreases serum DPP IV activity within 3-5 days and the immunochemotherapy-induced decreases in serum DPP IV activity are significantly and inversely related to treatment-induced increases in severity of depression and signs of activation of the IRS.

Adult↗

PubMed2000

In vitro immunization of patient T cells with autologous bone marrow antigen presenting cells pulsed with tumor lysates.

Presentation of cell-associated antigen to T cells is a critical event in the initiation of an anti-tumor immune response but it appears to often be deficient or limiting. Here we report an experimental system for stimulation of human T lymphocytes using autologous antigen presenting cells (APCs) and autologous tumor cells. Two types of APCs were prepared from human bone marrow: MC and DC. MC were produced by using GM-CSF and SCF. DC were obtained with the same cytokines plus IL-4. DC and MC were generated in parallel from the same patients and their phenotypes and capacities to prime T lymphocytes were analyzed and compared. MC were CD14+, CD1a-, CD33+ and HLA-DR+. Two populations of DC were defined: immature DC were uniformly CD1a-; mature DC expressed CD1a, CD80, CD86, HLA-DR, CD54 and CD58 but lacked surface CD14. Stimulation of autologous T lymphocytes was studied by measuring their proliferation and cytotoxic function. In more than 80% of our experiments the proliferation of autologous T lymphocytes cocultured with APC pulsed or not with tumor cell lysates was higher than that of T cells cultured alone. DC were more effective than MC in stimulating proliferation of lymphocytes. The capacity of a patient's autologous bone marrow-derived APC to stimulate T cells when exposed to autologous tumor cell lysates suggest that such antigen-exposed APC may be useful in specific anti-tumor immunotherapy protocols.

Antigen-Presenting Cells↗

PubMed2000

Renal synthesis of leukaemia inhibitory factor (LIF), under normal and inflammatory conditions.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine that is particularly involved in nephrogenesis and repair of the extracellular matrix. Transgenic mice overexpressing LIF have mesangial proliferative glomerulonephritis. Also, during local inflammatory reactions, such as kidney graft rejection or urinary tract infections, urinary LIF excretion is enhanced. The aim of the study therefore was to study LIF production by normal and inflammatory diseased kidneys (glomerulonephritis or graft rejection), maintained in short cultures. To determine the responsibility of the kidney itself in LIF synthesis, we measured LIF secretion into the culture supernatants of human mesangial or renal tubular epithelial cells. Fragments from diseased kidneys, whether grafts or not, released more LIF than normal human kidney fragments, mesangial or renal tubular epithelial cells. However, LIF production was delayed in renal transplants compared to glomerulonephritic samples taken from untreated patients. In every case, LIF production was enhanced by interleukin 1beta (IL-1beta) and inhibited by IL-4 or dexamethasone, except in two severe rejection episodes. So, LIF appeared to respond to pro- and anti-inflammatory stimuli, in vitro and in vivo. Considering its biological effects, LIF could play a role in inflammatory renal diseases.

Animals↗

PubMed1998

Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network.

Multidrug resistance (MDR) associated with overexpression of the MDR1 gene and of its product, P-glycoprotein (Pgp), plays an important role in limiting cancer treatment efficacy. Many studies have investigated Pgp expression in clinical samples of hematological malignancies but failed to give definitive conclusion on its usefulness. One convenient method for fluorescent detection of Pgp in malignant cells is flow cytometry which however gives variable results from a laboratory to another one, partly due to the lack of a reference method rigorously tested. The purpose of this technical note is to describe each step of a reference flow cytometric method. The guidelines for sample handling, staining and analysis have been established both for Pgp detection with monoclonal antibodies directed against extracellular epitopes (MRK16, UIC2 and 4E3), and for Pgp functional activity measurement with Rhodamine 123 as a fluorescent probe. Both methods have been validated on cultured cell lines and clinical samples by 12 laboratories of the French Drug Resistance Network. This cross-validated multicentric study points out crucial steps for the accuracy and reproducibility of the results, like cell viability, data analysis and expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Sequential production of leukaemia inhibitory factor by blood cell culture in patients with ARDS.

OBJECTIVE: Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine integrated in cytokine networks and its concentration has been shown to be elevated in bronchoalveolar lavage fluid of patients with the acute respiratory distress syndrome (ARDS). The aim of our study was to evaluate the production of LIF by culturing blood cells from patients with ARDS. PATIENTS: 8 patients with ARDS, 8 patients with pneumonia and 5 healthy subjects. MEASUREMENTS AND RESULTS: The blood samples were taken on day 1 after onset of ARDS. LIF was measured, in the cell-free supernatant, with an enzyme-linked immunosorbent assay after 24 h, 48 h and 72 h of blood cell culture. LIF was detectable in some patients in the ARDS group: at i) at 24 h and 48 h: in 2 patients ii) at 72 h in 4/5 patients (140 +/- 231 pg/ml). Only in the 4 patients in whom LIF was measured at 72 h was ARDS associated with the multiple organ dysfunction syndrome. Furthermore, among the 5 patients with ARDS who subsequently died, 4 had a detectable LIF. CONCLUSIONS: We have observed that LIF was produced only in ARDS, but not in all patients. The production of LIF seems to be a good indicator of the severity of ARDS. These preliminary results must be confirmed by a larger study.

Aged↗

PubMed1998

Effect of PGE2 on the cell surface molecule expression in PMA treated thymocytes.

PGE2 is produced by cells of the thymic microenvironment. The effects of PGE2 are mediated by cAMP through binding to its intracellular receptor protein kinase A (PKA). Phorbol 12-myristate 13-acetate (PMA) is known to modulate CD molecule expression on thymocytes, probably through activation of protein kinase C (PKC). We have hypothesized that cross-talk between these two signalling pathways may affect modulation of the CD molecules on the cell surface of thymocytes. For this purpose, we compare the effects of PMA alone or combined with PGE2 on CD3, CD4 and CD8 expression on mouse thymocytes by flow-cytometric analysis. PMA treatment almost completely abolished CD4 expression and slightly decreased CD3 and CD8 expression. PGE2 alone did not change the CD3, CD4 and CD8 molecule expression. Combined with PMA, PGE2 can overcome the decrease induced by PMA of the CD3 expression and partially reduced the disappearance of the CD4 molecule. On the other hand PGE2 accelerated the loss of CD8 molecule expression. These events occurred only in CD4+ CD8+ immature thymocytes. An analogue of cAMP (dibutyryl cAMP) mimics the effect of PGE2, but not Br-cGMP. This differential regulation by PGE2 of the CD molecule expression on immature thymocytes may provide additional evidence on the role of PGE2 during the process of thymic differentiation.

Animals↗

PubMed1998

Inhibition of graft-versus-host reaction by treatment of immature thymocytes with eicosanoids.

A model of in vivo secondary graft-versus-host (GVH) was used to appraise the behaviour of allostimulated eicosanoid-treated thymocytes. During the first step of the experiment P1 immature parental thymocytes were preincubated in the presence of P2 allogenic mature lymphocytes and eicosanoids (either PGE2 or LTB4). During the second step, the preincubated cells were injected intravenously into P1xP2 F1 irradiated hybrids and the in vivo mixed lymphocyte reaction assessed by 3H-thymidine uptake by the cells harboured in the spleen of the host. We also investigated the production of cytotoxic cells. We observed that PGE2 and LTB4 both influence the behaviour of immature thymocytes and that, in the context of allostimulation such as GVH, they induce a state of tolerance. The data are in agreement with a model of involvement of eicosanoids in education of thymocytes within the thymic organ.

Animals↗

PubMed1998

Leukemia inhibitory factor: part of a large ingathering family.

Leukemia Inhibitory Factor (LIF) has a wide variety of biological activities. It regulates the differentiation of embryonic stem cells, neural cells, osteoblasts, adipocytes, hepatocytes and kidney epithelial cells. It also triggers the proliferation of myoblasts, primordial germ cells and some endothelial cells. Many of these biological functions parallel those of interleukin-6, Oncostatin M, ciliary neurotrophic factor, interleukin-11 and cardiotrophin-1. These structurally related cytokines also share subunits of their receptors which could partially explain the redundancy in this system of soluble mediators. In vivo LIF proves important in regulating the inflammatory response by fine tuning of the delicate balance of at least four systems in the body, namely the immune, the hematopoietic, the nervous and the endocrine systems. Although we are far from its therapeutic applications, the fast increasing knowledge in this field may bring new insights for the understanding of the cytokine biology in general.

Animals↗

PubMed1997

A monoclonal antibody based elisa for quantitation of human leukaemia inhibitory factor.

The authors report on the development of a new sandwich enzyme-linked immunoabsorbent assay (ELISA) for the quantitation of the human cytokine leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) with high accuracy and sensitivity (23 pg/ml), in less than 5 h and in various biological fluids. The antibodies used in this assay were raised against recombinant glycosylated LIF expressed in vivo following inoculation of recombinant vaccinia viruses, and screened with the biologically active cytokine in a flow cytometry assay using cells expressing a membrane-bound form of LIF. Furthermore, this home-made assay was compared with two commercially available ELISA kits. The results led to the conclusion that these three assays are far from being equivalent between each other, in terms of sensitivity towards non-glycosylated vs glycosylated LIF. Two major parameters must be incriminated: the glycosylation status of the LIF molecule used as the calibrator, and the binding characteristics of the monoclonal antibodies used to set up these assays toward LIF derived from Escherichia coli or from eukaryotic cells. This points out the importance of these parameters for the design of ELISAs meant for the quantitation of glycosylated cytokines in biological fluids.

Animals↗

PubMed1997

In vitro production of human antigen presenting cells issued from bone marrow of patients with cancer.

In the prospect of producing autologous antigen presenting cells (APC) to actively immunize patients with cancer against their own tumor we were interested in the in vitro generation of MC and/or dendritic cells. We observed that the best yielding in CD14+ cells was obtained by adding SCF and GM-CSF into RPMI 1640 completed medium and by using Teflon bags as culture-containers. The others growth factors tested (LIF, IL3 and M-CSF) were useless in term of production of macrophages. After a month of culture we usually obtained an average of 80% of CD14, CD33, CD64, CD11a, CD11b, CD11c and HLA-DR positive cells expressing the MGG staining phenotype of MC. For DC the best association of growth factors combined GM-CSF, IL-4 and SCF. Hence we could obtained at least 60% of CD1a+, CD14-, CD54+, CD58+, CD80+ and HLA DR+ dendritic cells.

Antigen-Presenting Cells↗

PubMed1997

Refolding of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) from urea.

The insoluble inclusion bodies of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) was solubilized in 8 M urea on the unfolding transitions, and several factors on the aggregate formation were indirectly analyzed for the refolding of gp 190 sol DAF. Results indicate that the refolding yield can be considerably increased at lowering concentration of the unfolding protein, a little soluble protein with the slow refolding appears in the process of the aggregate formation and the concentration of the denaturant must be down to a minimum level for its refolding.

Glycerol↗

PubMed1995

Heterogeneity of murine adherent interleukin-2-activated killer cells. Differential effect of prostaglandin E2 and forskolin.

We have studied the relationship between cytotoxic activity, size and granularity of murine interleukin-2-activated adherent killer cells issued from spleen cells cultured with high levels of IL-2. The effects of prostaglandin E2 (PGE2) and forskolin upon these cells were assessed. All adherent spleen cells obtained after 5 days of culture were large granular lymphocytes but presented a heterogeneity in size and granularity. After fractionation on a discontinuous-density Percoll gradient, four cellular subpopulations were isolated. Fluorescence-activated cell sorting analysis showed that cells of the lightest fraction (F1) were the largest, while the cells found in the heaviest fraction (F4) were much more granular than the cells collected in the two intermediate fractions (F2 and F3). The serine esterases level was higher in F4 than in unfractionated cells and diminished to about 40% in cells of fractions F2 and F3, which expressed a cytotoxic activity against YAC-1 cells higher than that in unfractionated cells or in F1 or F4, which presented the lowest cytotoxic activity. When AK cells were cultured for 48 h in the presence of either PGE2 or forskolin, which induce an intracellular increase of cAMP, we observed that PGE2 (1 microM) inhibited the cytotoxic activity, but surprisingly forskolin (2 microM) exerted a stimulating effect on the induction of cytotoxic activity. After fractionation on a discontinuous Percoll gradient we observed the same cellular distribution among PGE2 or forskolin-treated or -untreated cells, but PGE2 induced an increase of size and granularity. This effect of PGE2 was more potent on the cells collected in F4. However this variation of granularity was not associated with any variation in the serine esterase level. The cytotoxic activity of PGE2- or forskolin-treated cells did not present any significant variation relative to the control for cells collected in F2 and F3; on the other hand, forskolin-treated cells collected in F4 showed a significantly higher cytotoxicity than did the corresponding untreated or PGE2-treated cells.

Animals↗

PubMed1994

One-year enzyme-linked immunosorbent assay follow-up of human interleukin for Da cells/leukemia inhibitory factor in blood and urine of 22 kidney transplant recipients.

The cytokine human interleukin for Da cells/leukemia inhibitory factor (HILDA/LIF) exerts multiple biological effects in vitro. In mice, high circulating levels of HILDA/LIF induce a wide range of pathophysiological events, some of them closely involved with immunological and inflammatory responses. Using a sandwich ELISA recognizing the natural human HILDA/LIF molecule with a threshold of 50 pg/ml in urine and 150 pg/ml in plasma, we monitored the urine and plasma HILDA/LIF levels of 22 patients in their first year after a kidney transplant. HILDA/LIF urine excretion is increased during acute rejection, and infections also trigger heavy HILDA/LIF plasma concentrations or urine excretion. In addition, this study raises the question of HILDA/LIF involvement in post-kidney-transplant phenomena such as hypercalcemia, osteoporosis, or the reversal of anemia.

Adolescent↗

PubMed1994

Radiobiological features of acute myeloblastic leukemia: comparison of self-renewal versus terminally differentiated populations.

PURPOSE: To evaluate the radiosensitivity of self-renewing progenitor cells in acute myeloblastic leukemia (AML), we have compared the radiosensitivity of the cells grown either in methylcellulose alone for 7 days, or first in suspension culture for 7 days before being plated in methylcellulose. Methylcellulose selects for terminal-dividing cells and suspension cultures have been developed because they allow self-renewal to occur: The exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. METHODS AND MATERIALS: Cells were harvested from previously untreated leukemic human bone marrows. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical, and immunophenotypic analysis, and by the use of growth factors that did not stimulate the growth of T-lymphocytes. The cell-cycle distribution of the blasts was analyzed by flow cytometry and was comparable for all samples. The irradiation was performed with gamma-photons at a dose-rate of 0.05 Gy/min, similar to the clinical conditions used in our institution for total body irradiation (TBI). RESULTS: The culture methods selected aggressive leukemias. There were large variations of the individual radiosensitivity whatever culture method was used. The progenitor cells capable of self-renewal were more radiosensitive than terminal dividing cells. In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. In these two cases, the best fit for the data was the linear quadratic model (survival = e-alpha D-beta D2) with alpha/beta values of 1.49 Gy and 3.12 Gy, respectively. CONCLUSION: The very low values of alpha/beta suggest a reduced antileukemic effect in case of fractionated TBI, and may lead to more reliable screening methods to determine the most appropriate technique for radiation ablation of bone marrow prior to bone marrow transplantation (BMT).

Adolescent↗