Biomedical subjects

M Guenounou

Publications and source records attributed to M Guenounou.

At least 19 recordsLinked to original sources

PubMed2004

Dysregulation of IL-2 and IL-8 production in circulating T lymphocytes from young cystic fibrosis patients.

It is well documented that patients with cystic fibrosis (CF) are unable to clear persistent airway infections in spite of strong local inflammation, suggesting a dysregulation of immunity in CF. We and others have reported previously that T lymphocytes may play a prominent role in this immune imbalance. In the present work, we compared the reactivity of CD3+ T cells obtained from young CF patients in stable clinical conditions (n = 10, aged 9-16.5 years) to age-matched healthy subjects (n = 6, aged 9-13.5 years). Intracellular levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-8 and IL-10 were determined by flow cytometry after whole blood culture. The data identified T lymphocyte subsets producing either low levels (M1) or high levels (M2) of cytokine under steady-state conditions. We found that the production of IFN-gamma and IL-10 by T lymphocytes was similar between young CF patients and healthy subjects. In contrast, after 4 h of activation with PMA and ionomycin, the percentage of T cells producing high levels of IL-2 (M2) was greater in CF patients (P = 0.02). Moreover, T cells from CF patients produced lower levels of IL-8, before and after activation (P = 0.007). We conclude that a systemic immune imbalance is present in young CF patients, even when clinically stable. This disorder is characterized by the capability of circulating T lymphocytes to produce low levels of IL-8 and by the emergence of more numerous T cells producing high levels of IL-2. This imbalance may contribute to immune dysregulation in CF.

Adolescent↗

PubMed2003

Activation of the cellular mitogen-activated protein kinase pathways ERK, P38 and JNK during Toxoplasma gondii invasion.

Host cell invasion is essential for the pathogenicity of the obligate intracellular protozoan parasite Toxoplasma gondii. In the present study, we evaluated the ability of T. gondii tachyzoites to trigger phosphorylation of the different mitogen-activated protein kinases (MAPK) in human monocytic cells THP1. Kinetic experiments show that the peak of extracellular-signal-regulated kinase (ERK1/2), P38 and cjun-NH2 terminal kinase (JNKs) phosphorylation occurs between 10 and 60 min. The use of specific inhibitors of ERK1/2, P38 and JNK1/2 phosphorylation indicates the specificity of MAPKs phosphorylation during invasion. Signaling through cellular and parasite mitogen-activated protein (MAP) kinase pathways appears to be critical for T. gondii invasion.

Animals↗

PubMed2002

Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells.

The coccidian Toxoplasma gondiiis an obligate intracellular parasite which can infect all cell types. Among the cytokines elicited by the immune response to Toxoplasma, tumor necrosis factor alpha (TNF-alpha) acts synergistically with gamma interferon (IFN-gamma) and plays a major role in host cell resistance. We have previously reported that TNF-alpha production induced by IFN-gamma/LPS decreases after T. gondii infection of human myelomonocytic THP-1 cells. Here, we investigated the regulation of TNF-alpha and its specific receptors during T. gondii infection of THP-1 cells. We found that TNF-alpha production was regulated at a post-transcriptional level and that TNF receptor expression was regulated at a pretranscriptional level. The TNF receptor I shedding and the fall in TNF-alpha levels observed after T. gondii infection would thus be induced by a parasite component with serine protease activity. These findings indicate that T. gondii participates not only in controlling the cytotoxic effects of TNF-alpha during the infection process, but also in signal transduction mediated mainly by TNF receptor I.

Animals↗

PubMed2001

Effect of hydroxyapatite sintering temperature on intracellular ionic concentrations of monocytes: a TEM-cryo-X-ray microanalysis study.

Hydroxyapatite used as bone replacement can lead to particle release in the implantation site. These particles interact with monocytes, which are the first immune cells to colonize the implant and an inflammatory site. Thanks to cryo-X-ray microanalysis, we can observe cells in a state close to the physiological one and we have access to diffusible ions. We paid particular attention to the potassium-to-sodium ratio, which is one of the best viability criteria. We used this method to study the interaction between three hydroxyapatite particles treated at three different temperatures (not treated, treated at 600 degrees C and 1180 degrees C), and monocytes. In the culture condition, the hydroxyapatite treated at 1180 degrees C underwent the least dissolution. We demonstrate that monocytes were altered by the three hydroxyapatite particles. The hydroxyapatite particules treated at 600 degrees C were found to be more toxic.

Bone Substitutes↗

PubMed2001

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

Journal Article↗

PubMed2000

Involvement of tumor necrosis factor-alpha during infection of human monocytic cells by Toxoplasma gondii.

Although the involvement of tumor necrosis factor-alpha (TNF-alpha) during Toxoplasma gondii infection has largely been described in mouse models, only a few studies in human models have been reported. We demonstrated the role of TNF-alpha during the process of invasion by T. gondii in human monocytic cells. Cotreatment of cells with interferon-gamma (IFN-gamma) and lipolysaccharide (LPS) during T. gondii infection induced TNF-alpha production and decreased the number of parasitized cells (invasion index). Neutralization of the production of TNF-alpha using a specific monoclonal antibody or pentoxifylline enhanced the invasion index. The relationship between TNF-alpha production and protection of monocytic cells against T. gondii invasion was confirmed by treatment of infected cultures with exogenous TNF-alpha. Thus, in contrast to the results obtained in murine models but in accordance with those observed in other human models, the present study shows for the first time in human monocytic cells that T. gondii does not induce any TNF-alpha secretion and inhibits TNF-alpha production induced by IFN-gamma/LPS.

Animals↗

PubMed2000

Implication of interleukin-4 in wound healing.

Interleukin-4 (IL-4) is a pleiotropic cytokine that is able to activate connective tissue cells and stimulate the accumulation of extracellular matrix macromolecules. In this report, the expression of IL-4 in normal wound healing was studied by immunohistochemistry. The effects of exogenous IL-4 or IL-4 antisense oligonucleotides administration were also studied in mouse experimental wounds. IL-4 expression was detected in the lower dermis below the wound as early as Day 1 after wounding. IL-4 expression was maximal by Day 4, then decreased progressively, and completely disappeared by Day 21 after wounding. Topical administration of IL-4 on experimental wounds in mice significantly accelerated the rate of healing, whereas IL-4 antisense oligonucleotides significantly inhibited healing. These results demonstrate that IL-4 may be implicated in normal wound healing.

Animals↗

PubMed2000

Comparison of clarithromycin-sensitive and clarithromycin-resistant Mycobacterium avium strains isolated from AIDS patients during therapy regimens including clarithromycin.

OBJECTIVES: Sixteen Mycobacterium avium strains were isolated from the blood of eight AIDS patients over a period of months. All the patients were on combination therapies including clarithromycin, and all had treatment failure and relapses of M.avium bacteremia. Paired clarithromycin-sensitive and resistant M.avium strains isolated at the beginning of treatment and at the first relapse of bacteremia were compared. METHODS: The M.avium isolates were identified after hybridization with DNA probes specific for M.avium rRNA and typed epidemiologically with random amplified polymorphic DNA analyses using three arbitrary primers. The rate of intracellular cell entry or the tumour necrosis factor alpha induction by the M.avium isolates were studied in human monocytes and J774 cells. RESULTS: When the M.avium isolates were hybridized with the rRNA probes, we obtained lower hybridization values with clarithromycin-resistant isolates than with clarithromycin-sensitive isolates. This appeared to be due to smaller amounts of rRNA available for hybridization than to mutation of the 23S rRNA sequences in clarithromycin-resistant strains. The RAPD analyses showed that the clarithromycin-resistant isolates were clonally related to the clarithromycin-sensitive strains in six of the eight patients. The other two patients had a RAPD profile, suggesting a re-infection and/or polyclonal infection. The M.avium isolates obtained on day 0 and after the emergence of resistance to clarithromycin did not differ in terms of their intracellular entry rate, or in terms of tumour necrosis factor alpha induction. CONCLUSIONS: We infer that M.avium strains isolated during bacteraemic relapses on combination therapies including clarithromycin are epidemiologically related to the initial strain and do not show changes in the rate of intracellular cell entry and in terms of tumour necrosis factor alpha induction. Re-infections and/or polyclonal infections however, although less frequent, can also occur.

AIDS-Related Opportunistic Infections↗

PubMed2000

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) are essentially dependent on visceral fat amount in type 2 diabetic patients.

TNF-alpha is considered as one of the potential determinants of insulin resistance. However several data suggest that TNF-alpha expression itself, could be modulated by the degree of adiposity and/or plasma insulin levels. To clarify the determinants of plasma TNF-alpha levels in type 2 diabetes mellitus, we studied the impact of intensive insulin treatment on plasma TNF-alpha levels in 16 type 2 diabetic subjects with failure to oral antidiabetic medication (HbA1c: 10.8 +/- 1.2 %). Furthermore, we analyzed the relationship between plasma TNF-alpha levels and total or regional body fat measurements using anthropometry, bienergetic absorptiometry and computed tomography in a cohort of 33 caucasian obese type 2 diabetic subjects (BMI: 32.2 +/- 4.4 kg/m(2) ). The plasma TNF-alpha level was neither affected by plasma glucose level variations nor intensive insulin treatment despite a 37 % decrease in daily insulin needs at the end of insulin therapy (total duration: 11.5 +/- 2.0 days). The plasma TNF-alpha level was similar in men and women and unrelated to age, fasting glycemia or HbA1c. A relationship was highlighted with BMI (r =0.39, p <0.02), but not with total fat mass. This relationship was only dependent on the intra-abdominal fat mass amount as assessed by the waist-to-hip circumference ratio (r =0.52, p <0.01) and the visceral adipose tissue area (r =0.56, p <0. 01). These results show that plasma TNF-alpha levels are essentially dependent on visceral fat amount, thus suggesting that TNF-alpha could be one of the factors mediating insulin resistance and cardiovascular risk in obese type 2 diabetic patients.

Absorptiometry, Photon↗

PubMed1999

HIV-1 gp160 decreases the K+ voltage-gated current from Jurkat E6.1 T cells by up-phosphorylation.

HIV-1 gp120/gp160 is known to disturb the activity of p56lck, protein kinase C (PKC) and Ca2+ homeostasis in T lymphocytes. We found that gp160 decreases the Kv1.3 current of Jurkat E6.1 cells probably by increasing the PKC-dependent phosphorylation of Kv channel protein after 5 days. This decrease is dose-dependent. In contrast, gp160 did not decrease the Kv1.3 current of the JCaM1.6 cell line, a p56(lck)-defective Jurkat cell line. This shows that p56lck was at the beginning of the events which induced the Kv1.3 current decrease. As a consequence of this decrease, Jurkat E6.1 cells were depolarized and exhibited a volume increase.

HIV Envelope Protein gp160↗

PubMed1999

Expression of IL-17 in human memory CD45RO+ T lymphocytes and its regulation by protein kinase A pathway.

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8(+)45RO+T cells and CD4(+)45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8(+)T cells required accessory signals since culture of ionomycin/PMA-activated CD8(+)45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4(+)T cell population seems to be more independent. IL-17 and interferon gamma(IFN-gamma) mRNA were both inhibited in the presence of PGE2 or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4(+)and CD8(+)T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2 or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2 induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2 may be more efficient in blocking both IL-17 and IFN-gamma expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4(+) and CD8(+)T cells during the immune and inflammatory processes.

Bucladesine↗

PubMed1998

Regulation of IL-17, IFN-gamma and IL-10 in human CD8(+) T cells by cyclic AMP-dependent signal transduction pathway.

In the present study, the expression of interleukin 17 (IL-17) by human CD8(+) T lymphocytes and its regulation following PKA activation was determined and compared with that of interferon gamma (IFN-gamma) and IL-10. IL-17 mRNA was highly expressed in human CD8(+) T lymphocytes at least at the same level than in CD4(+) T cells that were isolated from peripheral blood mononuclear cells (PBMC). Expression of IL-17 mRNA in CD8(+) T cell was induced by prior activation of PBMC for 18 h with Ca2+ ionophore and phorbol myristate acetate (PMA). Furthermore, our results clearly showed that CD8(+) T cells are sensitive to elevation of cAMP and PKA activation pathway. Data demonstrated a significant inhibition of IL-17 as well as of IFN-gamma mRNA expression in CD8(+) T cells isolated from activated PBMC cultured in the presence of either dibutyryl cAMP (db-cAMP) or PGE2. In contrast, IL-10 mRNA expression was strongly enhanced in the same experimental conditions. The differential expression of IL-10 and IFN-gamma production in CD8(+) T cells was also observed at the protein level as it was measured by a double immunofluorescence technique and flow cytometry analysis. Taken together, these results provide evidence that human CD8(+) T cells are also the source of massive expression of IL-17, and that PKA plays a prominent role in the switch of CD8(+) T cells to a Th2 like profile and an inhibition of IL-17 expression, thus suggesting that the activation of cAMP signal transduction pathway may have consequences for the relative role of CD8(+) T cells in the immune and inflammatory process.

CD8-Positive T-Lymphocytes↗

PubMed1998

Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro.

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Adolescent↗

PubMed1998

Interferon-gamma signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii.

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii. In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii. The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-gamma. However, IFN-gamma treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-gamma altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-gamma directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-gamma may be involved in mediating their protective signals.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Pyrimethamine-sulfadoxine treatment of congenital toxoplasmosis: follow-up of 78 cases between 1980 and 1997. Reims Toxoplasmosis Group.

UNLABELLED: The purpose of this study was to determine the clinical and immunological outcome of 78 children with congenital toxoplasmosis treated with the pyrimethamine-sulfadoxine combination between 1980 and 1997. METHODS: Children were divided into 3 groups according to the initial duration of treatment (always including folinic acid, 5 mg/week by mouth), as follows: pyrimethamine (1.25 mg/kg every 15 d) + sulfadoxine (25 mg/kg every 15 d) for 12 months (Group 1, 47 children), or for 24 months, with or without prenatal therapy (respectively, Group 2, 19 children, and Group 3, 12 children). RESULTS: Chorioretinitis occurred in 23% of these 78 children. Four children had unilateral blindness, 1 had mild epileptic fits and 1 had psychomotor retardation. The lowest rate of sequelae were in Groups 2 and 3. Immunological rebounds, generally without clinical repercussions, occurred frequently (90% of cases on average) during, or more often after therapy, regardless of the treatment duration. Treatment was always well tolerated. CONCLUSIONS: Our current treatment strategy for congenital toxoplasmosis consists of a 24-month course of pyrimethamine-sulfadoxine (Fansidar) combined with folinic acid (Lederfoline). If the prenatal diagnosis is positive, we also prescribe this treatment to the mother until delivery. This combination offers satisfactory compliance, adequate serum concentrations, and good preventive efficacy.

Adolescent↗

PubMed1997

Does human toxoplasmosis involve an imbalance in T1/T2 cytokines?

The T1 (interferon-gamma, interleukin-12, interleukin-2) and T2 (interleukin-4, interleukin-10, interleukin-6) cytokine groups constitute two polar responses of the immune system. The T1 group is a predominantly cellular response, while the T2 group response is mainly humoral. The hypothesis forwarded here links these subgroups of induced cytokines to the various clinical forms of human toxoplasmosis. Ocular toxoplasmosis in immunocompetent patients could be attributed to a T1 hyper-response, whereas congenital toxoplasmosis, toxoplasmic encephalitis (in immunodeficient patients) and active chronic toxoplasmosis (with persistent lymphadenophathy) would be characterized by a predominantly T2 response. Confirmation that this kind of immunological imbalance effectively underlies the various clinical forms of toxoplasmosis would open the way for a new range of treatments based on immunomodulation.

Animals↗

PubMed1997

Differential regulation of IFN-gamma, IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP-dependent signal transduction pathway.

Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signalling pathways. In the present work, we attempted to clarify the role of cAMP on interferon-gamma (IFN-gamma), interleukin (IL)-10, IL-4 and IL-13 expression as well as on the inducible nitric oxide synthase (iNOS) expression. Treatment of phytohaemagglutinin (PHA)/phorbol 12-myristate 13-acetate (PMA)-activated Jurkat cells with either dibutyryl-cyclic adenosine monophosphate (cAMP) or pentoxifylline induced a strong inhibition of IFN-gamma mRNA expression as measured by reverse transcription (RT)-polymerase chain reaction (PCR), without affecting IL-10 expression. Both cholera toxin and prostaglandin E2 (PGE2) induced a strong inhibition of IFN-gamma mRNA expression, whereas IL-10 mRNA expression was significantly enhanced. This differential regulation of IFN-gamma and IL-10 expression was related to intracellular cAMP concentration. IL-13 and IL-4 mRNA expressions were not inhibited. We developed a new method based on immunofluorescence for intracellular cytokine detection followed by optical and computerized image processing, and our results showed that IFN-gamma protein was strongly inhibited when cells were treated with PGE2 or dibutyryl (db)-cAMP, whereas IL-10 protein was enhanced. This suggests that cAMP exerts its action at both the transcriptional and protein levels. iNOS mRNA expression was markedly elevated in the presence of PGE2. The generation of nitric oxide using sodium nitroprusside (SNP) induced a dramatic decrease of IFN-gamma, while IL-10 was enhanced; and conversely the inhibition of iNOS activity using 1-NG-monomethyl arginine (1-NMMA) induced a clear inhibition of IL-10 and IL-4, while IFN-gamma was enhanced. These results provide evidence that the protein kinase A (PKA) activation pathway plays a prominent role in the balance between the type 1 and type 2 cytokine profile in PHA/PMA-activated Jurkat cells. Data also suggest that iNOS expression is under the control of PKA activation, and that NO seems to be able to assume the polarization of activated T cells to the type 2 profile.

Cholera Toxin↗

PubMed1996

Inhibition of human TNF alpha and LT in cell-free extracts and in cell culture by antisense oligonucleotides.

Antisense oligonucleotides (ODN), complementary to mRNA of human tumor necrosis factor alpha (TNF alpha) and lymphotoxin (LT) were tested for their ability to inhibit TNFs. TNFs production was studied in cell-free systems including wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). All ODN were effective in WGE at low concentration (0.2 microM), except those targeted to the 3' region of TNF alpha mRNA. A short ODN complementary to a common region between TNF alpha and LT inhibited both TNFs. In contrast, high ODN concentration (50 microM) was needed to inhibit LT mRNA translation in RRL, whereas no clear inhibition of TNF alpha was observed unless RNase H was added to the translation mixture. ODN effects on TNFs production by stimulated cell line in culture were also investigated. Three ODN-one located in the 5'-untranslated region, one spanning the AUG initiation codon and one downstream of this AUG-were the most effective sequences to decrease TNF alpha production. Two ODN targeted to the AUG initiation codon of LT were also able to inhibit its production. In conclusion we confirm the role of RNase H in cell free systems, and we found that there is no correlation between ODN efficiency in a cell-free system nor in cell culture. Efficient ODN could be used for in vitro investigation of the role of TNF alpha and LT in mechanism in which they are involved.

Animals↗