Functional responsiveness of in vitro-aged human neutrophils.

Elimination of apoptotic neutrophils by macrophages is as a major step for the resolution of inflammation. However, the fate and the cellular functionality of neutrophils aged in the absence of macrophages are not well documented. Herein, freshly isolated human neutrophils were aged for several days in vitro and then stimulated with agonists for determining their cell responsiveness. In vitro-aged neutrophils were still able to generate reactive oxygen species after 48 h, exert phagocytosis after 72 h, and increase their adhesion onto a cell substratum after 48 h. These data demonstrate that a portion of neutrophils cultivated for several days in vitro are still able to exert biological functions. This opens the possibility that, during inflammation, neutrophils may still respond to agonists, a condition that is likely to occur in vivo when they are not efficiently eliminated by efferocytosis.

Human neutrophils are targets to paracoccin, a lectin expressed by Paracoccidioides brasiliensis.

OBJECTIVE AND DESIGN: Paracoccin (PCN), a lectin expressed by Paracoccidioides brasiliensis (Pb), is known to exert activities on the fungal biology, as well as different immune cells of myeloid origin. The aim of this study was to investigate the direct interaction of the recombinant form of the lectin (rPCN) with neutrophils, a neglected area. MATERIALS OR SUBJECTS: Freshly isolated human neutrophils from healthy donors were used. TREATMENT: Neutrophils were incubated with rPCN in vitro. METHODS: After the treatment, the production of reactive oxygen species (ROS), DNA release, IL-8, TNF, IFN-γ, IL-10, IL-12p40, TGF-ß and IL-1ß production, fungicidal ability, apoptosis and de novo protein synthesis was determined. RESULTS: rPCN was found to induce ROS production as well as DNA release. Using the ROS inhibitor, diphenyleneiodium, both ROS production and DNA release were significantly inhibited. In addition, rPCN was found to induce IL-8 and IL1-ß production, inhibit apoptosis and induce de novo protein synthesis. Addition of cycloheximide, a protein synthesis inhibitor, drastically reversed the antiapoptotic effect of rPCN. Finally, the ability to kill Pb yeasts by human neutrophils was significantly increased after rPCN stimulation. CONCLUSIONS: rPCN can alter the biology of human neutrophils increasing their fungicidal ability. Moreover, the ability of rPCN to increase DNA release and to induce suppression of neutrophil apoptosis occurs by a ROS- and de novo protein synthesis-dependent mechanism, respectively.

Mechanism involved in interleukin-21-induced phagocytosis in human monocytes and macrophages.

The interleukin (IL)-21/IL-21 receptor (R) is a promising system to be exploited for the development of therapeutic strategies. Although the biological activities of IL-21 and its cell signalling events have been largely studied in immunocytes, its interaction with human monocytes and macrophages have been neglected. Previously, we reported that IL-21 enhances Fc gamma receptor (FcRγ)-mediated phagocytosis in human monocytes and in human monocyte-derived macrophages (HMDM) and identified Syk as a novel molecular target of IL-21. Here, we elucidate further how IL-21 promotes phagocytosis in these cells. Unlike its ability to enhance phagocytosis of opsonized sheep red blood cells (SRBCs), IL-21 did not promote phagocytosis of Escherichia coli and zymosan by monocytes and did not alter the cell surface expression of CD16, CD32 and CD64. In HMDM, IL-21 was found to enhance phagocytosis of zymosan. In addition, we found that IL-21 activates p38, protein kinase B (Akt), signal transducer and activator of transcription (STAT)-1 and STAT-3 in monocytes and HMDM. Using a pharmacological approach, we demonstrate that IL-21 enhances phagocytosis by activating some mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)-Akt and Janus kinase (JAK)-STAT pathways. These results obtained in human monocytes and macrophages have to be considered for a better exploitation of the IL-21/IL-21R system for therapeutic purposes.

Nanoparticles enhance the ability of human neutrophils to exert phagocytosis by a Syk-dependent mechanism.

BACKGROUND: Some reports indicate that NPs are ingested by cells via different mechanisms, including phagocytosis. In contrast, the direct role of NPs on the phagocytic process is not well documented. The aim of this study was to determine if titanium dioxide (TiO(2)), zinc oxide (ZnO) and cerium dioxide (CeO(2)) NPs, could alter the ability of neutrophils to exert phagocytosis. METHODS: Freshly isolated human neutrophils were incubated with NPs and their ability to phagocytose opsonized sheep red blood cells (SRBCs) or fluorescent latex beads (LBs) was assessed by optical and fluorescence microscopy, respectively. Syk activation was assessed by western blot experiments and a pharmacological approach with piceatannol, a Syk inhibitor, was used to determine its role in NPs-induced neutrophils. The cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) was used as a positive control. RESULTS: All tested NPs enhanced the ability of neutrophil to phagocytose SRBCs and LBs. Syk was activated in NPs-induced neutrophils as evidenced by its increased tyrosine phosphorylation level vs controls and the ability of NPs-induced phagocytosis was reversed by piceatannol. CONCLUSIONS: We found that the tested NPs enhanced phagocytosis, although at different degree, and this occurred by a Syk-dependent mechanism. GENERAL SIGNIFICANCE: This is the first study demonstrating that NPs, by themselves, can directly enhance FcR-mediated (opsonized SRBCs) and complement-mediated (LBs) phagocytosis. Moreover, as part of their mode of action, we determined that NPs can act similarly to GM-CSF leading to Syk activation involved in phagocytosis. This has to be taken under consideration for future nanobiology and nanomedicine studies.

Pregnancy-related listeriosis in France, 1984 to 2011, with a focus on 606 cases from 1999 to 2011.

This study describes trends in the incidence of pregnancy-related listeriosis in France between 1984 and 2011, and presents the major characteristics of 606 cases reported between 1999 and 2011 to the French Institute for Public Health Surveillance through the mandatory notification system. The incidence of pregnancy-related listeriosis decreased by a factor of 12 from 1984 to 2011. This reduction was a result of progressive implementation of specific Listeria monocytogenes control measures in food production. A lower incidence of pregnancy-related listeriosis was observed in regions with a lower prevalence of toxoplasmosis. Given that dietary recommendations in pregnancy target both toxoplasmosis and listeriosis prevention, we suppose that recommendations may have been delivered and followed more frequently in these regions. Cases reported between 1999 and 2011 (n=606) were classified as maternal infections with ongoing pregnancy (n=89, 15%), fetal loss (n=166, 27%), or live-born neonatal listeriosis (n=351, 58%). The majority of live-born neonatal listeriosis cases (n=216, 64%) were preterm births (22­36 weeks of gestation), of whom 14% (n=30) were extremely preterm births (22­27 weeks of gestation). Eighty per cent of mothers reported having eaten high risk food during pregnancy. A better awareness of dietary recommendations in pregnant women is therefore necessary.

Severe kerion Celsi caused by Trichophyton quinckeanum: Severe kerion Celsi due to Trichophyton quinckeanum.

We report a severe case of kerion Celsi of the scalp in a previously healthy 13-year-old girl due to Trichophyton quinckeanum, an emerging dermatophyte species in Europe. The species was definitely identified by DNA sequencing and the patient was successfully treated by oral terbinafine for 6 weeks. Kerion Celsi is a severe inflammatory form of tinea capitis, which is characterised by a purulent discharge and alopecia [1]. It typically occurs in children infected with zoophilic dermatophytes, such as Trichophyton mentagrophytes, and an increasing number of cases caused by other Trichophyton species has recently been reported [2]. Herein we report a severe case of kerion Celsi of the scalp caused by the emerging species Trichophyton quinckeanum, which was successfully treated by oral antifungal.

Evidence that polyhydroxylated C60 fullerenes (fullerenols) amplify the effect of lipopolysaccharides to induce rapid leukocyte infiltration in vivo.

Fullerenols C60(OH) have therapeutic potential, but there is debate regarding their toxicity. Here, we tested the hypothesis that C60(OH)n possesses a pro-inflammatory effect in vivo. Kinetic and dose-dependent experiments performed with the murine air pouch model of acute inflammation revealed that, unlike TiO2 used as a positive control in this model, C60(OH)n NPs were not pro-inflammatory in CD-1, C57BL/6, and BALB/c mice. However, after 3 h of treatment, C60(OH)n NPs were found to amplify the effect of lipopolysaccharides (LPS) causing a rapid leukocyte influx in which the major cells observed are neutrophils. The use of an antibody array assay to detect different analytes simultaneously indicates that the amplification effect is, at least partially, explained by an increased local production of several cytokines/chemokines in the exudates, including the pro-inflammatory cytokine IL-6. Using an ELISA to quantify the amount of IL-6 produced into air pouch exudates, we demonstrated that C60(OH)n increases the LPS-induced local production of this cytokine. Therefore, although C60(OH)n NPs alone do not exert proinflammatory activity under certain conditions, they can act in concert with other agents to cause inflammation, a situation that is likely to occur in vivo.

Inhibition of neutrophil infiltration by a malleable protein matrix of lactic acid bacteria-fermented whey proteins in vivo.

OBJECTIVE: A novel nutraceutical ingredient, the Malleable Protein Matrix (MPM), has previously demonstrated a significant anti-inflammatory effect in a systemic inflammatory disease model, comparable to conventional drugs. The objective of this study was to investigate the potential anti-inflammatory effects of MPM on neutrophil infiltration in vivo, phagocytosis activity as well as cytokine and chemokine production. METHODS: Groups of ten C57BL\6J mice received water or MPM per os for a period of 2 weeks prior to the creation of a murine air pouch. The subsequent neutrophil recruitment and activities were characterized following lipopolysaccharide injection. RESULTS: In the water control group, the number of recruited cells was 1.8X10(7) cells/pouch, which was reduced to 9X10(6) cells/pouch with oral MPM consumption, representing an inhibition of 50% of infiltrating leukocytes. A considerable reduction in the cytokine and chemokine production, mostly TNFalpha, IL-1beta and IL-6 production in the MPM-treated group, suggested an inhibition of the mediators responsible for leukocyte extravasation. On the other hand, MPM consumption had no effect on neutrophil phagocytosis activity. CONCLUSION: MPM administration demonstrates a significant reduction of neutrophil infiltration associated with an inhibition of cytokine and chemokine production. The air pouch model shares similarities with in vivo characteristics of rheumatoid arthritis and neutrophilic diseases, both of which would benefit from this 50% inhibition of neutrophil infiltration induced by MPM.

Activation of human eosinophils with palladium nanoparticles (Pd NPs): importance of the actin cytoskeleton in Pd NPs-induced cellular adhesion.

Palladium (Pd) is known to be released into the environment in the fine and ultrafine (at the nanoscale) airborne particle fractions mainly from automobile catalytic converters leading to an increase human exposure to this noble metal. It was reported that Pd can induce allergic reactions in individuals exposed to it via different ways. Some studies reported an increased number of eosinophils into airways following NP exposure in vivo in rodent models of allergies and inflammation. Knowing the importance of eosinophils in allergies, asthma and other lung diseases, it is surprising to observe that the direct effect of Pd at the nanoscale in eosinophils has been poorly documented. The aim of this study was to determine how Pd NPs will affect the biology of human eosinophils. Characterization of Pd NPs by dynamic light scattering indicates the presence of some aggregates when suspended in diverse solutions used here for the different experiments. Pd NPs did not significantly induce cell necrosis and apoptosis in eosinophils (0.5-150µg/ml) as assessed by trypan blue exclusion assay, flow cytometry after staining with FITC-annexin V and propidium iodide and by morphological observations by optical microscopy. PD NPs, unlike the positive controls, did not induce reactive oxygen species (ROS) but were found to target the actin cytoskeleton, since actin was differently re-located intracellularly when compared to untreated cells as determined by fluorescence microscopy. Clearly, Pd NPs were found to increase adhesion of eosinophils onto human endothelial EA.hy926 cells. Using cytochalasin D, a cell-permeable and potent inhibitor of actin polymerization, this ability to increase adhesion was drastically reversed. Our results indicate that Pd NPs can target the cytoskeleton and increase the adhesion of human eosinophils by an actin-dependent mechanism. These findings show that human eosinophils can be activated by Pd NPs emphasizing the importance of fully investigating how these NPs could alter the biology of human cells involved in allergies, asthma and other lung diseases as well as in various other inflammatory disorders.

Patient and bacterial determinants involved in symptomatic urinary tract infection caused by Escherichia coli with and without bacteraemia.

Risk-factors for bacteraemia were determined in a case-control study of patients with Escherichia coli urinary tract infection. Cases were defined as patients with E. coli urinary source bacteraemia, and controls were chosen from among patients with E. coli urinary tract infection without bacteraemia. Patient characteristics were collected prospectively and the bacterial traits were determined. The phylogenetic background and virulence factors of E. coli isolates did not differ between cases and controls. In multivariate analysis, being female and having a urinary catheter were significantly less prevalent among patients with urinary source bacteraemia than among patients with uncomplicated urinary tract infection.

[Optoelectronic gait analysis after metatarsophalangeal arthrodesis of the hallux: fifteen cases]. / Analyse opto-électronique de la marche après arthrodèse métatarso-phalangienne de l'hallux: à propos de 15 cas.

PURPOSE OF THE STUDY: Degeneration of the metatarsophalangeal joint of the hallux is a frequent secondary lesion of the first ray subsequent to hallux valgus. Different surgical techniques have been proposed for cure, including metatarsophalangeal arthrodesis. Joint fusion relieves pain but sacrifices joint motion. The purpose of this work was to assess changes observed in gait after metatarsophalangeal arthrodesis of the hallux using a three-dimensional optoelectronic system. MATERIAL AND METHODS: Gait analysis was performed with a Vicon 3D system with five cameras and two AMTI force platforms in twelve patients who had undergone metatarsophalangeal arthrodesis more than six months earlier. The kinetic and kinematic curves and ground reaction forces were analyzed. Changes in the gait cycle and any compensations observed in the talocrural and interphalangeal joints were noted in the three dimensions. Wilcoxon test for paired series was applied for the statistical analysis. RESULTS: The general gait parameters and kinetic and kinematic values were not modified (excepting a non-significant decline in maximal dorsiflexion of the ankle joint). There was a significant decrease in propulsion force in the anteroposterior and vertical planes, with significantly later heal lift-off and systematic displacement of ground reaction forces anterior to the metatarsophalangeal joint on the arthrodesis side. Reflectors positioned on the distal extremity of the hallux demonstrated that the essential part of compensation occurred at the level of the interphalangeal joint. DISCUSSION: Gait analysis after tibiotalar arthrodesis has been widely reported in the literature. The consequence of joint fusion on the rear foot and/or the torsion couple have also been studied. However, to our knowledge, there has been only one report using a different methodology devoted to metatarsophalaneal arthrodesis of the hallux. In this study, only step length and interphalangeal moment as well as ankle force were found to be decreased. Function of the interphalangeal joint was not assessed. The Vicon system enabled an adapted study of gait after metatarsophalangeal arthrodesis. This method offers several perspectives: study of the effect of the position of the arthrodesis in the sagittal plane on gait, changes over time in interphalangeal joint motion, or consequences of fusion on walking up and down stairs. CONCLUSION: Metatarsophalangeal arthrodesis of the hallux does not modify general gait parameters nor the kinetic and kinematic values. Compensation is achieved via the interphalangeal joint.

Evaluation of frequency of paediatric oral liquid medication dosing errors by caregivers: amoxicillin and josamycin.

OBJECTIVE: To study reconstitution and preparation dosing errors of liquid oral medications given by caregivers to children. METHODS: A prospective observational study was carried out in the departments of general paediatrics and emergency paediatrics at the Robert-Debré Children's University Hospital. An interview with caregivers involved (1) practical reconstitution and preparation of an oral liquid medication from a prescription drawn at random (amoxicillin (Clamoxyl, dosing spoon) or josamycin (Josacine, dose-weight pipette)) and (2) a questionnaire about their use. RESULTS: One hundred caregivers were included. Clamoxyl and Josacine were incorrectly reconstituted in 46% (23/50) and 56% (28/50) of cases, respectively, with a risk of underdosing of Clamoxyl (16/23) and overdosing of Josacine (23/28). Dose preparation with the dosing spoon was incorrect in 56% of cases, and in 10% of cases with the dose-weight pipette. Female sex, native French speaker, and age were significantly associated with correct reconstitution. Male sex and medication were significantly associated with correct preparation. CONCLUSIONS: This study highlights the high incidence of errors made by caregivers in reconstituting and preparing doses of these liquid oral medicines, which are associated with considerable risks of over- and underdosing. Factors associated with these errors have been identified which could help health professionals to optimise their strategy for educating families about the use of liquid oral medications and the need to check that they understand these instructions.

Effects of interleukin-13 on human neutrophil functions.

It was recently shown that interleukin-13 (IL-13) induces the expression and release of the IL-1 decoy receptor (type II) in human neutrophils along with the production of the IL-1 receptor antagonist. In the present study we focused on further studying the modulatory effects of IL-13 on these cells. We found that recombinant human IL-13 (rhIL-13) induces cellular morphological changes in neutrophils that are typical of activated cells. Furthermore, rhIL-13 was shown to increase tyrosine phosphorylation of several neutrophil proteins. We also demonstrate that this cytokine stimulates de novo RNA synthesis in a concentration-dependent fashion as measured by [5-3H]uridine incorporation and that rhIL-13 induces the synthesis of several neutrophil proteins according to high-resolution two-dimensional gel electrophoresis of metabolically [35S]-labeled cells. We observed that the IL-8 levels in the external milieu of IL-13-stimulated cells was almost fivefold increased when compared with control cells. Finally, we observed that rhIL-13 has no significant effect on phagocytosis and apoptosis. Taken together, our results demonstrate that IL-13 is a modulator of several human neutrophil functions. This leads us to conclude that the modulatory role of IL-13 on human neutrophils, a potentially important anti-inflammatory cytokine, is more complex than previously believed. Furthermore, because we show that the synthesis of several as yet unidentified proteins was up-regulated by IL-13, our findings open new avenues of research on the effects of this cytokine on human neutrophils.

Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo.

Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 microM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis.

The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolically 35S-labeled human neutrophils when used at low concentrations (< 100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) approaches, we found that VAA-I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA-I was also found to reverse the delaying effect of GM-CSF on neutrophil apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In contrast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself and does not alter the GM-CSF-induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA-1077, and z-VAD-FMK, only the latter (inhibitor of caspases-1, -3, -4, and -7) was found to inhibit VAA-I-induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 +/- 1.3 to 54 +/- 3.2% (n=4). Furthermore, we confirm that caspases are involved in VAA-I-induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase-3-dependent. Such degradation was reversed by the z-VAD-FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA-I is a caspase-dependent mechanism that does not involve tyrosine phosphorylation events, G-proteins, PKCs, and PLA2. In addition, we conclude that at least caspase-3 is involved. Correlation between VAA-I-induced neutrophil apoptosis and VAA-I-induced inhibition of de novo protein synthesis is discussed.

Peanut shell colitis.

A 40-year-old physician experienced abdominal pain, loose stools, hematochezia, and anal discomfort with defecation associated with the daily consumption of 15 to 30 whole peanuts, including the shells. Thorough evaluation revealed only nonspecific colitis of the distal portion of the sigmoid colon and inflamed hemorrhoids. Discontinuation of whole peanut ingestion was associated with symptomatic, endoscopic, and histological resolution. In this patient, undigested peanut shells seem to have caused a nonspecific colitis, perhaps as the result of mechanical abrasion of the colonic mucosa.

Emphysematous cholecystitis following cardiopulmonary resuscitation.

Emphysematous cholecystitis developed in a 65-year-old man 24 hours following resuscitation from cardiac arrest. Our findings in this case support the importance of ischemia in this disease process.

Second messengers involved in the regulation of corticotropin-releasing hormone mRNA and peptide in cultured rat fetal hypothalamic primary cultures.

Using primary cultures of dispersed rat fetal hypothalami, we studied the effect of forskolin and the phorbol ester 12-o-tetradecanoyl phorbol 13-acetate, activators of protein kinase A and C, respectively, on corticotropin-releasing hormone (CRH) regulation. CRH mRNA accumulation and peptide release were stimulated by both agents, indicating that the protein kinase A and protein kinase C messenger systems are involved in the regulation of CRH gene expression and are functional in hypothalamic neurons isolated from fetal brain.

Adrenocorticotropin-cortisol axis abnormalities in hemodialysis patients.

The ACTH-cortisol axis was studied in 15 hemodialysis patients. Basal plasma cortisol concentrations were found to be elevated and ACTH to be in the high normal range. Cortisol responded normally to exogenous ACTH, but neither cortisol nor ACTH were suppressed in response to oral dexamethasone. 11-Deoxycortisol and ACTH concentrations did not rise normally in response to either oral or iv metyrapone. We conclude that standard testing of the ACTH-cortisol axis in dialysis patients yields results suggesting Cushing's syndrome.

Mechanisms in hypokalemia: clinical correlation.

Hypokalemia is seen most often with the use of diuretics and in patients with emesis. Other common clinical settings in which it may be significant include corticosteroid therapy, antibiotic usage, diarrhea, diabetic ketoacidosis, or psychiatric illness. Occasionally the cause may be obscure. In such situations the determination of urine potassium and arterial pH may prove helpful. Subclassification of hypokalemia into such categories as "acidosis", "alkalosis", "extra-renal", or "renal" loss is then possible. The cases discussed demonstrate the utilization of these methods to define the etiology and to understand the pathophysiology in hypokalemia.