Search

About 0.1% of the sera in human pathology produce a peculiar, cytoplasmic, non-organ- and non-species-specific fluorescence. This may easily be differentiated from the already described anti-organelle antibodies and, more particularly, from the mitochondrial antibodies of primary biliary cirrhosis. Should rat tissues he used in the immunofluorescence test, fluorescence predominates over the first two portions of the renal proximal tubules (P1 and P2) and the mucous neck cells of the stomach. This pattern may be attributed to mitochondria, and in particular to their inner membranes by fluorescent staining of the ellipsoid region of the rods and cones of the eyes, and by absorption with purified organelles. To distinguish this antibody from the already described mitochondrial antibodies, this one will be called mitochondrial antibody number 5 (M5). The seven carriers of this antibody suffer from systemic lupus erythematosus or autoimmune haemolytic anaemia. In these cases no diseases of the liver were observed, contrary to other classical mitochondrial antibodies. [ABSTRACT FROM AUTHOR]

Macrolide antibiotics are taken up and concentrated by host cells, particularly phagocytes, and are likely candidates to modify cell functions. In this study, we extended our previous work concerning the effect of three 14-membered-ring macrolides (dirithromycin, erythromycin and erythromycylamine) on human neutrophil exocytosis, and found that three other erythromycin A derivatives (roxithromycin, clarithromycin and the azalide, azithromycin) also triggered neutrophil degranulation in a time- and concentration-dependent manner. After 30 min of incubation, the correlation coefficients for concentration-dependence for roxithromycin were 0.885, 0.739 and 0.750 (P < 0.005) and for clarithromycin were 0.795, 0.599, 0.733 (P < 0.005), respectively, for lysozyme, β-glucuronidase and lactoferrin release. Although the underlying mechanism was not elucidated, these and previous data suggest that intracellular accumulation is a prerequisite. Furthermore, comparison of the characteristics of macrolide-induced exocytosis with those of exocytosis triggered by the synthetic chemotactic stimulus FMLP suggested that different mechanisms are involved. In keeping with this possibility, we showed that combined treatment (macrolides plus FMLP) resulted in totally additive exocytosis of azurophilic but not specific granules. The clinical relevance of our data remains to be ascertained. [ABSTRACT FROM PUBLISHER]

Dirithromycin, a new semisynthetic 14-membered-ring macrolide was avidly concentrated by human neutrophils in a time- but not concentration-dependent manner with mean cellular/extracellular, concentration ratios (C/E) of 9 within the first 5 min and up to 47 at 120 min. Erythromycylamine, the hydrolysis product of dirithromycin, was concentrated significantly less by neutrophils, reaching C/E values of 4 and 19 (at 5 and 120 min). A point of interest was the interindividual variability in the antibiotic uptake kinetics; in particular, 7 out of 47 neutrophil samples from different healthy volunteers displayed very slow uptake of both drugs (C/E values at 30 min: dirithromycin, 5·8; erythromycylamine, 4·6). The reason(s) for this is unknown. The uptake of both drugs was decreased at acidic pH and increased at basic pH. Chloroquine, an antimatarial drug which is concentrated in and alkalinizes azurophilic granules, reduced uptake by half. Metabolic inhibitors (2–4 dinitrophenol, sodium fluoride, potassium cyanide and sodium azide) did not impair the uptake of either drug but, interestingly, ouabain, an inhibitor of membrane Na/K ATPase activity, impaired uptake by about 30% Competitive inhibitors of some transport systems identified on neutrophil membrane (nucleosides, D-glucose and various aminoacids) did not alter the uptake of either drug. Dirithromycin and to a lesser extent, erythromycylamine, reached intracellular concentrations much higher than those required to inhibit the growth of sensitive microorganisms. Although the mechanism of uptake is not clear, one interesting hypothesis involves trapping by protonation into acidic compartments of neutrophils. [ABSTRACT FROM PUBLISHER]

Polymorphonuclear neutrophils (PMN) play a prominent role in the host response to infectious diseases. One major bactericidal mechanism used by these cells is the production of reactive oxygen species during what is referred to as the oxidative burst. However, excessive oxidant generation can also be involved in cell and tissue damage associated with severe inflammatory reactions. Macrolide antibiotics are able to penetrate and concentrate within phagocytes and have been successfully used to treat infections due to facultative intracellular pathogens. However, intracellular accumulation of macrolides with possible alkalinization of cellular compartments may interfere with normal cell function. In-vitro and ex-vivo data suggest that macrolides affect various phagocytic functions. This paper presents an overview of the published data concerning the modulation of neutrophil function by macrolides. Preliminary data concerning the in-vitro modulation of the neutrophil oxidative burst by dirithromydn and its metabolite, erythromycylamine, are also discussed. [ABSTRACT FROM PUBLISHER]

Josamycin and erythromycin have been compared for their in-vitro interaction with bactericidal killing by human neutrophils. The mechanism of this interaction was studied in two ways. First, the target organisms (Staphylococcus aureus and Pseudomonas aeruginosa) were incubated for 60 min with josamycin, erythromycin or control buffer prior to use in a human polymorphonuclear neutrophil (PMN) killing assay. Second the macrolides were added directly to acellular killing systems mimicking those acting inside the phagolysosome; oxygen-independent systems were obtained from a crude granule extract of PMN and oxygen-dependent systems consisted either of a mixture of xanthine plus xanthine oxidase or of a solution of H2O2. Whereas josamycin-pretreated P. aeruginosa were twice as sensitive to killing by PMN than were control cells, this was not the case for S. aureus. Both oxidant generating systems were more effective in destroying S. aureus in the presence of josamycin (3 and 30 mg/l). Erythromycin showed a similar synergy but only with the xanthine plus xanthine oxidase system. This synergy was observed with neither of the O2-independent systems for S. aureus, nor with any acellular system for P. aeruginosa. These data suggest that at least two kinds of mechanism may explain the bactericidal synergy observed between macrolides and PMN. The first (for macrolide-resistant species such as P. aeruginosa) could be due to alterations in the bacteria by the antibiotics, while the second (for macrolide-sensitive species such as S. aureus) could be based upon an as yet unexplained transformation of the molecules by reactive oxygen species into more "toxic" forms. These differences between josamycin and erythromycin could arise from differences in their chemical structure. [ABSTRACT FROM AUTHOR]

Cefodizime, a 2-amino-thiazolyl cephalosporin, is reported to display in-vitro, ex-vivo and in-vivo immunomodulatory properties; in particular, it enhances the survival of mice infected with cefodizime-resistant pathogens. We have used an in-vitro model to assess the indirect effect of this drug (compared with other cephalosporins) on the neutrophil (PMN) oxidative response. Pseudomonas aeruginosa was employed as the bacterial target for cefodizime and cefotaxime (MICs > 128 mg/l), cefsulodin (MIC 16 mg/l) and ceftazidime (MIC 32 mg/l). After overnight growth in the presence of subinhibitory concentrations of each drug (10 mg/l), the altered filamentousP. aeruginosa induced a stronger oxidative response of PMN than untreated control bacteria. For all cephalosporins this was related to alterations of bacterial structure leading to increased deposits of antibodies and/or complement. Furthermore, increased non-opsonin dependent stimulation of the PMN oxidative burst was obtained; the strongest response was observed with cefodizime-treated P. aeruginosa in the case of low responder PMN, which displayed a deficient response after stimulation by control bacteria. The possibility that cefodizime could enhance this PMN function in opsonin-deficient patients requires further investigation. [ABSTRACT FROM PUBLISHER]

Immunomodulation by antibacterial agents shows promise as a novel strategy in the treatment of infectious diseases. Cefodizime, a new oxi-imino-amino-2-thiazolyl cephalosporin, is a particularly good candidate in this context. In-vivo models of experimental infections show that prophylactic administration of cefodizime increases the survival of some strains of mice after challenge with Toxoplasma gondii or Candida albicans; its curative effect in infections due to members of the Enterobacteriaceae is better than that expected from in-vitro MIC determinations relative to other third-generation cephalosporins; this effect is even more marked in immuno-compromised animals. Data obtained both in vivo and ex vivo show that cefodizime enhances various immune parameters such as phagocyte function, B lymphocyte responsiveness and delayed hypersensitivity; it may restore natural killer (NK) and phagocyte activity, as well as interleukin 1 (IL-1) and interferon production, in immunocompromised patients and animals. The in-vitro effects of this drug include enhancement of phagocyte bactericidal activity and alteration of bacterial virulence factors. The chemical basis for these various immunomodulatory properties is related to the thio-thiazolyl side-chain at position 3 of the cephem nucleus. To date, the mechanisms underlying the immunomodulatory properties of cefodizime have not been identified clearly, but it is likely that it interferes at different levels of specific and non-specific immune defences. [ABSTRACT FROM PUBLISHER]

Cefodizime is a 2-aminothiazolyl cephalosporin for parenteral use. Cefodizime has a bisubstituted thiothiazole moiety in position 3 of the cephem nucleus. The presence of this moiety does not alter the in-vitro antibacterial activity, or safety in animal studies, which are similar to those of cefotaxime, but results in an apparent long elimination half-life in rodents and dogs, and in novel immunological properties. [ABSTRACT FROM PUBLISHER]

We have compared the in-vitro interactions of quinine and cinchonine, two alkaloids from cinchona bark, with human neutrophil functions. Although these molecules are structurally similar, they induced a quantitatively different depressive effect on neutrophil chemotaxis and oxidative response. Quinine produced the strongest effect at concentrations as low as 10 mg/l, which may be achievable in serum during therapeutic use of this compound. The depression induced by cinchonine was noticeable only at 100 mg/l. Chemotaxis was decreased by about 25% (formyl-methionyl-leucyl-phenylalanine) or 39% (serum) for quinine (100 mg/l) only if a constant concentration of the drug was maintained during the assay while cinchonine had no effect on this PMN function. The greatest impairment was observed for the PMN oxidative burst: this was dose-dependent whatever the stimulus used (phorbol-myristate-acetate or opsonized zymosan). After 30 min incubation in the presence of the drugs, the zymosan-induced chemiluminescence response was decreased by 96% and by 67% with quinine, 100 and 10 mg/l, respectively, and by 62% with cinchonine 100 mg/l. The myeloperoxidase-mediated iodination of PMN was reduced by 100% and 46% with quinine, 100 and 10 mg/l, respectively, whereas cinchonine decreased this function by about 95% at 100 mg/l and 14% at 10 mg/l. Superoxide anion generation was impaired by 94% (quinine 100 mg/l) or 45% (cinchonine 100 mg/l). The relevance to the clinical situation and the possible mechanisms of such effects are discussed. [ABSTRACT FROM AUTHOR]

We have compared the in-vitro interactions of quinine and cinchonine, two alkaloids from cinchona bark, with human neutrophil functions. Although these molecules are structurally similar, they induced a quantitatively different depressive effect on neutrophil chemotaxis and oxidative response. Quinine produced the strongest effect at concentrations as low as 10 mg/1, which may be achievable in serum during therapeutic use of this compound. The depression induced by cinchonine was noticeable only at 100 mg/1. Chemotaxis was decreased by about 25% (formyl-methionyl-leucyl-phenylalanine) or 39% (serum) for quinine (100 mg/l) only if a constant concentration of the drug was maintained during the assay while cinchonine had no effect on this PMN function. The greatest impairment was observed for the PMN oxidative burst: this was dose-dependent whatever the stimulus used (phorbol-myristate-acetate or opsonized zymosan). After 30 min incubation in the presence of the drugs, the zymosan-induced chemiluminescence response was decreased by 96% and by 67% with quinine, 100 and 10 mg/1, respectively, and by 62% with cinchonine l00 mg/1. The myeloperoxidase-mediated iodination of PMN was reduced by 100% and 46% with quinine, 100 and 10 mg/1, respectively, whereas cinchonine decreased this function by about 95% at 100 mg/1 and 14% at 10 mg/1. Superoxide anion generation was impaired by 94% (quinine 100 mg/1) or 45% (cinchonine 100 mg/1). The relevance to the clinical situation and the possible mechanisms of such effects are discussed. [ABSTRACT FROM PUBLISHER]

Josamycin, a 16-membered ring macrolide is concentrated up to 20-fold in phagocytic cells compared with serum. We have studied the in-vitro interaction of this drug with human neutrophils (PMN) bactericidal function by using two strains resistant to this antibiotic, Pseudomonas aeruginosa and Klebsiella pneumoniae, and a sensitive one, Staphylococcus aureus 209P. It was shown that josamycin-pretreated adherent PMN displayed an increased phagocytic activity (about 30 to 40%) for S. aureus or K. pneumoniae, mainly due to the recruitment of an additional phagocytizing subset of PMN. Furthermore, the bacterial killing was enhanced in josamycin-treated PMN in a dose-dependent manner for K. pneumoniae (60-130% increase in the range of concentration 0.1-25 mg/l) and independently of the dose for S. aureus (about 425-460% increase for josamycin 0.1-10 mg/l). P. aeruginosa killing by whole blood was also significantly increased in the presence of 10 and 1 mg/l of josamycin. Other PMN functions were not much altered by josamycin except an enhancement of the formyl-methionyl-leucyl-phenylalanine-induced oxidative response. Chemotaxis was only increased by the presence of a high concentration (100 mg/l) of josamycin. These data suggest that the bactericidal synergy between PMN and josamycin could be related, partly at least, to a direct enhancing effect of josamycin on some PMN functions such as phagocytosis, chemotaxis and FMLP-induced chemiluminescence. On the other hand, alterations of bacteria, either inside the phagolysosome or in the extracellular medium, could lead to an enhanced susceptibility to the phagocytes' microbial mechanisms. [ABSTRACT FROM AUTHOR]

We have compared the in-vitro interaction of five macrolides (roxithromycin, erythromycin, spiramycin, oleandomycin and josamycin) with human neutrophils (PMN). Only roxithromycin strongly impaired the oxidative burst of PMN assessed by luminol amplified chemiluminescence, superoxide anion generation, and myeloperoxidase-mediated iodination of proteins. This effect was observed only for high concentrations of this drug (100 and 50 mg/l). Furthermore, the sensitivity of PMN to the depressive effect of roxithromycin permitted the definition of two kinds of PMN: in Highly Sensitive (HS)-PMN, the oxidative response was completely abolished while in Moderately Sensitive (MS)-PMN, a decreased, but yet measurable (20-50% of the control), response was obtained. The roxithromycin-induced depression of PMN was time-dependent and partly reversed by washing. Chemotaxis was also impaired by roxithromycin (100 mg/l) but phagocytosis of Klebsiella pneumoniae was unaltered even at high concentrations of the drug. Since roxithromycin displays the highest intracellular uptake, compared with the other macrolides assessed in this study, this could explain the results observed here. The relevance to the clinical situation needs further study. This effect of roxithromycin could be useful to control the inflammatory process associated in certain infectious diseases, in particular if high concentrations of the drug are obtained in tissues. [ABSTRACT FROM AUTHOR]

Ceftriaxone, a 2-aminothiazolyl cephalosporin does not alter human neutrophil (PMN) bactericidal function. However, low concentrations of ceftriaxone induce some bacterial strains to be more sensitive to PMN killing. We have studied the effect of a subinhibitory concentration of ceftriaxone (10 mg/l) on Pseudomonas aeruginosa (MIC greater than 128 mg/l). After an overnight exposure to this concentration of ceftriaxone, P. aeruginosa elongated into filaments. PMN killing of ceftriaxone-treated bacteria was better than killing of control bacteria. This enhanced killing was correlated with an increased sensitivity to oxygen-dependent bacterial killing. Furthermore, the altered bacteria induced a greater oxidative response of PMN which was independent of their chemiluminescence response after stimulation by control P. aeruginosa. This increased oxidative burst was attributable to both non-opsonodependent stimulation and to increased deposit of opsonins. [ABSTRACT FROM AUTHOR]

The enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxy-imino-amino- 2-thiazolyl cephalosporins, is linked to the cell stimulation of oxygen-dependent and oxygen-independent killing systems, respectively. Cefotaxime enhances both the killing and the oxidative response of neutrophils to opsonized particulate stimuli (bacteria for both activities and opsonized zymosan for the oxidative burst). These effects were not observed with non-opsonized particles (bacteria or zymosan) or soluble stimuli. On the contrary, cefodizime enhances killing of opsonized and non-opsonized bacteria by neutrophils regardless of treatment with phenylbutazone which blocks neutrophil oxidative metabolism. Cefodizime does not universally alter the oxidative burst induced by various stimuli, but has been shown to enhance the bactericidal activity of crude extracts of neutrophil granules. The data suggest that cefodizime and non O2-dependent killing systems of neutrophils cooperate in killing bacteria. [ABSTRACT FROM AUTHOR]

, cefotaxime and cefodizime enhanced significantly the bactericidal activity of human neutrophils against P 209 A, but not phagocytosis. The increase was about 150% for cefotaxime and 400% for cefodizime at concentrations as low as 1 mg/1. Furthermore, by two different techniques (NBT and cytochrome C reduction tests) cefotaxime but not cefodizime significantly enhanced superoxide anion production by zymosan-stimulated neutrophils. Other neutrophil functions (chemotaxis and myeloperoxidase-mediated iodination of proteins) were not significantly altered by either antibiotic, even at concentrations as high as 1000 mg/1. [ABSTRACT FROM PUBLISHER]

The sera of 322 patients with various connective tissue diseases were checked by immunofluorescence (IF) on rat tissue sections. Twelve sera containing ribosomal antibodies were selected by this procedure and their antigenic specificities were studied. It was possible to divide the sera into two groups: R sera, the IF pattern of which disappeared after RNase treatment of the tissue sections and the DR sera, still detectable after RNase treatment. In immunodiffusion (ID) these two groups were antigenically distinct but in each group complete identity of the precipitin lines was observed. The diversity of the ribosomal antigens involved was shown. The prognostic value of these antibodies is discussed. [ABSTRACT FROM AUTHOR]

Background: Advances in chromatography and mass spectrometry have allowed us to develop a novel technique for measuring intraprostatic hormone concentrations directly on prostate needle biopsies, rather than using traditional punch excision. This has significant clinical implications as intraprostatic dihydrotestosterone and testosterone levels could help monitor prostate growth, neoplasia and castration resistance., Methods: Patients undergoing radical cystoprostatectomy for bladder cancer were prospectively included. Each prostate specimen received one 90mg punch excision and six needle biopsies. Intraprostatic hormones were dosed through gas chromatography-mass spectrometry., Results: We included twenty patients, of which eleven were incidentally diagnosed with prostate cancer; four had ISUP 1 (20%) and seven had ISUP 2 (35%). The prostate biopsy technique was unable to obtain measures for testosterone, Delta-4-androsterone and androstenedione. Tissue concentrations of DHEA, DHT, E1 and E2 can be obtained with no significant difference from the reference established on a punch from a single biopsy core sample., Conclusions: Our study demonstrates that intraprostatic concentrations of DHEA, DHT, E1 and E2 can be measured without significant difference from the reference established on a single punch excision. This finding opens the way to research on the interactions between endocrinology and prostate oncogenesis and particularly on the mechanisms of resistance to hormone therapies in vivo., (Copyright © 2024 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Bioprinting technology offers unprecedented opportunities to construct in vitro tissue models that recapitulate the 3D morphology and functionality of native tissue. Yet, it remains difficult to obtain adequate functional readouts from such models. In particular, it is challenging to position sensors in desired locations within pre-fabricated 3D bioprinted structures. At the same time, bioprinting tissue directly onto a sensing device is not feasible due to interference with the printer head. As such, a multi-sensing platform inspired by origami that overcomes these challenges by "folding" around a separately fabricated 3D tissue structure is proposed, allowing for the insertion of electrodes into precise locations, which are custom-defined using computer-aided-design software. The multi-sensing origami platform (MSOP) can be connected to a commercial multi-electrode array (MEA) system for data-acquisition and processing. To demonstrate the platform, how integrated 3D MEA electrodes can record neuronal electrical activity in a 3D model of a neurovascular unit is shown. The MSOP also enables a microvascular endothelial network to be cultured separately and integrated with the 3D tissue structure. Accordingly, how impedance-based sensors in the platform can measure endothelial barrier function is shown. It is further demonstrated the device's versatility by using it to measure neuronal activity in brain organoids., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

We investigated using a custom NGS panel of 149 genes the mutational landscape of 64 consecutive adult patients with tyrosine kinase fusion-negative hypereosinophilia (HE)/hypereosinophilic syndrome (HES) harboring features suggestive of myeloid neoplasm. At least one mutation was reported in 50/64 (78%) patients (compared to 8/44 (18%) patients with idiopathic HE/HES/HE US used as controls; p < .001). Thirty-five patients (54%) had at least one mutation involving the JAK-STAT pathway, including STAT5B (n = 18, among which the hotspot N642H, n = 13), JAK1 (indels in exon 13, n = 5; V658F/L, n = 2), and JAK2 (V617F, n = 6; indels in exon 13, n = 2). Other previously undescribed somatic mutations were also found in JAK2, JAK1, STAT5B, and STAT5A, including three patients who shared the same STAT5A V707fs mutation and features consistent with primary polycythemia. Nearly all JAK-STAT mutations were preceded by (or associated with) myelodysplasia-related gene mutations, especially in RNA-splicing genes or chromatin modifiers. In multivariate analysis, neurologic involvement (hazard ratio [HR] 4.95 [1.87-13.13]; p = .001), anemia (HR 5.50 [2.24-13.49]; p < .001), and the presence of a high-risk mutation (as per the molecular international prognosis scoring system: HR 6.87 [2.39-19.72]; p < .001) were independently associated with impaired overall survival. While corticosteroids were ineffective in all treated JAK-STAT-mutated patients, ruxolitinib showed positive hematological responses including in STAT5A-mutated patients. These findings emphasize the usefulness of NGS for the workup of tyrosine kinase fusion-negative HE/HES patients and support the use of JAK inhibitors in this setting. Updated classifications could consider patients with JAK-STAT mutations and eosinophilia as a new "gene mutated-entity" that could be differentiated from CEL, NOS, and idiopathic HES., (© 2024 Wiley Periodicals LLC.)

Purpose: Post-thoracotomy pain syndrome (PTPS) and chronic postsurgical neuropathic pain (CPNP) were evaluated 4 months after thoracic surgery whether the approach was a posterolateral (PL) incision or the less invasive axillary (AX) one., Methods: Patients, 79 in each group, undergoing a thoracotomy between July 2014 and November 2015 were analyzed 4 months after surgery in this prospective monocentric cohort study., Results: More PL patients suffered PTPS (60.8% vs. 40.5%; p = 0.017) but CPNP was equally present (45.8% and 46.9% in the PL and AX groups). Patients with PTPS have more limited daily activities (p < 0.001) but a similar psychological disability (i.e., catastrophism). Patients with CPNP have an even greater limitation of daily activities (p = 0.007) and more catastrophism (p = 0.0002). Intensity of pain during mobilization of the homolateral shoulder at postoperative day 6 (OR = 1.40, CI 95% [1.13-1.75], p = 0.002); age (OR = 0.97 [0.94-1.00], p = 0.022), and presence of pain before surgery (OR = 2.22 [1.00-4.92], p = 0.049) are related to the occurrence of PTPS; while, height of hypoesthesia area on the breast line measured 6 days after surgery is the only factor related to that of CPNP (OR = 1.14 [1.01-1.30], p = 0.036)., Conclusion: Minimally invasive surgery was associated with less frequent PTPS, but with equal risk of CPNP. Pain before surgery and its postoperative intensity are associated with PTPS. This must lead to a more aggressive care of pain patients before surgery and of a better management of postoperative pain. CPNP can be forecasted according to the early postoperative height of hypoesthesia area on the breast line., (© 2023. The Author(s) under exclusive licence to Japanese Society of Anesthesiologists.)

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization., Competing Interests: The authors declare no conflicts of interest., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Traumatic brain injury (TBI), which is characterized by damage to the brain resulting from a sudden traumatic event, is a major cause of death and disability worldwide. It has short- and long-term effects, including neuroinflammation, cognitive deficits, and depression. TBI consists of multiple steps that may sometimes have opposing effects or mechanisms, making it challenging to investigate and translate new knowledge into effective therapies. In order to better understand and address the underlying mechanisms of TBI, we have developed an in vitro platform that allows dynamic simulation of TBI conditions by applying external magnetic forces to induce acceleration and deceleration injury, which is often observed in human TBI. Endothelial and neuron-like cells were successfully grown on magnetic gels and applied to the platform. Both cell types showed an instant response to the TBI model, but the endothelial cells were able to recover quickly-in contrast to the neuron-like cells. In conclusion, the presented in vitro model mimics the mechanical processes of acceleration/deceleration injury involved in TBI and will be a valuable resource for further research on brain injury., Competing Interests: No competing financial interests exist., (© Luise Schlotterose et al., 2023; Published by Mary Ann Liebert, Inc.)

Study Objective: Postoperative physical therapy and early mobilization are major elements for enhanced recovery after surgery. In contrast with supervised physical therapy sessions that can be monitored, self-mobilization is not easily quantifiable and has so far been estimated mainly through patient auto-reports. This study aimed to perform a comprehensive and objective evaluation of postoperative mobility., Design: Prospective observational study., Setting: Postoperative setting., Patients: Patients undergoing mini-invasive lung surgery., Interventions: Measurement of postoperative mobility during the first five postoperative days using an accelerometer (ActiGraph GT3X)., Measurements: The primary outcome was the number of daily steps. Secondary outcomes included physical activity duration and intensity, sedentary time, number of breaks in sedentary time, sedentary patterns, daily evaluation by physiotherapists, postoperative complications, and acceptability of wearing the accelerometer., Main Results: Sixty patients were included in the study, of whom 56 provided at least one day of valid accelerometry data. There was no significant change during the first four PODs concerning the number of daily steps nor the mean cadence. One-minute cadence peak, total activity counts, and duration of light-intensity physical activity increased over time (p = 0.032, p = 0.001 and p = 0.001, respectively). Sedentary patterns changed favorably over time, with a decrease in prolonged sedentary bouts (≥ 60 consecutive min) (p < 0.001), and an increase in shorter bouts (< 10 min) (p = 0.001). Similar results were observed when analysis was adjusted for the day of the week when the surgery took place. The median acceptability of wearing the accelerometer was excellent (median 10 [9-10] on a 10-point Likert scale). Three patients had major complications., Conclusions: Our findings suggest that daily steps may not be the only relevant indicator of early mobility following thoracic surgery and that accelerometry is suitable to follow patients' early postoperative activity., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) is a major health problem that affects millions of persons worldwide every year among all age groups, mainly young children, and elderly persons. It is the leading cause of death for children under the age of 16 and is highly correlated with a variety of neuronal disorders, such as epilepsy, and neurodegenerative disease, such as Alzheimer's disease or amyotrophic lateral sclerosis. Over the past few decades, our comprehension of the molecular pathway of TBI has improved, yet despite being a major public health issue, there is currently no U.S. Food and Drug Administration-approved treatment for TBI, and a gap remains between these advances and their application to the clinical treatment of TBI. One of the major hurdles for pushing TBI research forward is the accessibility of TBI models and tools. Most of the TBI models require costume-made, complex, and expensive equipment, which often requires special knowledge to operate. In this study, we present a modular, three-dimensional printed TBI induction device, which induces, by the pulse of a pressure shock, a TBI-like injury on any standard cell-culture tool. Moreover, we demonstrate that our device can be used on multiple systems and cell types and can induce repetitive TBIs, which is very common in clinical TBI. Further, we demonstrate that our platform can recapitulate the hallmarks of TBI, which include cell death, decrease in neuronal functionality, axonal swelling (for neurons), and increase permeability (for endothelium). In addition, in view of the continued discussion on the need, benefits, and ethics of the use of animals in scientific research, this in vitro , high-throughput platform will make TBI research more accessible to other labs that prefer to avoid the use of animals yet are interested in this field. We believe that this will enable us to push the field forward and facilitate/accelerate the availability of novel treatments., Competing Interests: No competing financial interests exist., (© Luise Schlotterose et al., 2023; Published by Mary Ann Liebert, Inc.)

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(ε-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes' alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle, as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle's physiological functions.

To understand the effect of mechanical stimulation on cell response, bone marrow stromal cells were cultured on electrospun scaffolds under two distinct mechanical conditions (static and dynamic). Comparison between initial and final mechanical and biological properties of the cell-constructs were conducted over 14 days for both culturing conditions. As a result, mechanically stimulated constructs, in contrast to their static counterparts, showed evident mechanical-induced cell orientation, an effective aligned collagen and tenomodulin extracellular matrix. This orientation provides clues on the importance of mechanical stimulation to induce a tendon-like differentiation. In addition, cell and collagen orientation lead to enhanced storage modulus observed under dynamic stimulation. Altogether mechanical stimulation lead to (a) cell and matrix orientation through the sense of the stretch and (b) a dominant elastic response in the cell-constructs with a minor contribution of the viscosity in the global mechanical behavior. Such a correlation could help in further studies to better understand the effect of mechanical stimulation in tissue engineering., (© 2021 Wiley Periodicals LLC.)

Tissue engineering is a promising approach to repair tendon and muscle when natural healing fails. Biohybrid constructs obtained after cells&rsquo; seeding and culture in dedicated scaffolds have indeed been considered as relevant tools for mimicking native tissue, leading to a better integration in vivo. They can also be employed to perform advanced in vitro studies to model the cell differentiation or regeneration processes. In this review, we report and analyze the different solutions proposed in literature, for the reconstruction of tendon, muscle, and the myotendinous junction. They classically rely on the three pillars of tissue engineering, i.e., cells, biomaterials and environment (both chemical and physical stimuli). We have chosen to present biomimetic or bioinspired strategies based on understanding of the native tissue structure/functions/properties of the tissue of interest. For each tissue, we sorted the relevant publications according to an increasing degree of complexity in the materials&rsquo; shape or manufacture. We present their biological and mechanical performances, observed in vitro and in vivo when available. Although there is no consensus for a gold standard technique to reconstruct these musculo-skeletal tissues, the reader can find different ways to progress in the field and to understand the recent history in the choice of materials, from collagen to polymer-based matrices.

It is well acknowledged that ansamycins display immunosuppressive and anti-inflammatory properties in vitro and in vivo. Rifalazil, a new ansamycin derivative, has not been studied in the context of inflammation. In particular, there are no data on the possible interference of rifalazil with oxidant production by phagocytes. We have compared the antioxidant properties of rifalazil to those of rifampin, a drug well known in this context, by using cellular and acellular oxidant-generating systems. Oxidant production by polymorphonuclear neutrophils was measured in terms of cytochrome c reduction, lucigenin-amplified chemiluminescence (Lu-ACL), and the 2',7'-dichlorofluorescin diacetate H2 (DCFDA-H2) technique (intracellular oxidant production). Rifalazil impaired O2- production in a concentration-dependent manner, with 50% inhibitory concentrations (IC50) (concentrations which inhibit 50% of the response) of 5.4 (30 and 60 min of incubation) and 6.4 (30 min) mg/liter, respectively, for phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. In agreement with the published fMLP-like activity of rifampin, the inhibitory effect of rifampin was significantly greater for fMLP (IC50 of 5.6 mg/liter) than for PMA (IC50 of 58 mg/liter) stimulation. Alteration of intracellular oxidant production was also observed with IC50 values similar to those obtained by the cytochrome assay. In addition, rifalazil and rifampin (> or = 25 mg/liter) scavenged O2-, as demonstrated by the acellular (hypoxanthine-xanthine oxidase) system. Interference with light detection systems was evidenced for both drugs by Lu-ACL. The clinical relevance of the antioxidant effect of rifalazil demonstrated in vitro, in particular its potential anti-inflammatory activity, requires further investigation.

Macrolides are accumulated in phagocytes, partially via an active transport system; the membrane carrier is not identified but many data indicate a link with the P-glycoprotein family which includes the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. We have used two epithelial cell lines which express either wild-type (N cells) or mutated (homozygous deltaF508) (F cells) CFTR to study the cellular accumulation of two macrolides (azithromycin and roxithromycin). Adherent cells were incubated with the radiolabeled drugs before extensive washings and counting. Azithromycin was better (about 2-fold) accumulated in F cells up to 60 min but then plateaued, whereas accumulation continued without saturation over 3 hours in N cells. Roxithromycin was also better (1.5-fold) accumulated in F cells at 15 and 30 min, but there were no differences at further incubation times. Macrolide efflux from loaded N and F cells, and the susceptibilities of the carrier systems (entry and efflux) to various pharmacologic agents were similar to those previously observed with phagocytes. These data suggest that the macrolide carriers (for entry and efflux) are not strictly specific for phagocytes and that the CFTR protein plays a role in macrolide uptake.

Interactions between antibacterial agents and polymorphonuclear neutrophils (PMNs) are a major focus of investigation. Owing to the variable drug susceptibility of PMNs from different individuals, in vitro studies require samples from large panels of healthy volunteers to reach statistical significance. Here, we used a phagocytic cell line, PLB-985, which can differentiate into mature PMNs in vitro, for the study of cellular interactions (drug uptake and antioxidant effects) of two macrolides (azithromycin and roxithromycin) and four ketolides [HMR 3004, HMR 3647 (telithromycin), HMR 3562 and HMR 3787]. The oxidative burst of differentiated (D) cells was inhibited by macrolides and ketolides. IC50% values (concentrations impairing the oxidative burst by 50%), determined after 30 min of incubation, were as follows for azithromycin, roxithromycin, HMR 3004, telithromycin, HMR 3562 and HMR 3787, respectively: 40, 39, 15, 23, 26, and 33 mg/l (fMLP stimulation) and 37, 86, 39, 43, 14, and 31 mg/l (PMA stimulation). These values were similar to those obtained with PMNs. Uptake of the two macrolides was significantly lower in non-differentiated (ND) cells than in D cells and PMNs. The cellular/extracellular (C/E) concentration ratios at 60 min for PMNs, D and ND PLB were respectively 67, 25 and 11 (roxithromycin) and 159, 137 and 48 (azithromycin). Ketolide uptake by ND-PLB was also significantly lower than that obtained with PMNs (C/E ratios at 60 min were about 75 versus 265 (HMR 3004), 36 vs 230 (telithromycin), 75 vs 235 (HMR 3562) and 20 vs 130 (HMR 3787). Although the active carrier system seemed to be present in ND cells, its activation pathway was not functional. Thus, the PLB-985 cell line is a good in vitro model for studying drug-PMN interactions. The use of ND and D cells may shed light on the nature and activation pathways of macrolide transport systems present on the PMN membrane.

HMR 3647 (telithromycin), a new ketolide, is active on intracellular pathogens. It was previously demonstrated that it inhibits superoxide anion production in a time- and concentration-dependent manner, at concentrations which inhibit 50% of the control response of about 55 microg/ml (5 min) to 30 microg/ml (30 min); these values are similar to those obtained with roxithromycin, a classical erythromycin A derivative. Here we investigated whether these drugs modified the bactericidal activity of human polymorphonuclear neutrophils (PMN) on four strains of Staphylococcus aureus with different profiles of susceptibility to macrolides and ketolides. We found that the main factor involved in killing was the antibacterial potency of the drugs, although combinations of antibiotics with PMN were slightly more active than each component used alone against two of the four strains. In addition, high concentrations of the drugs, which impaired the PMN oxidative burst, did not impair PMN bactericidal activity. Likewise, some cytokines which enhance PMN oxidative metabolism did not modify PMN bactericidal activity in the presence or absence of macrolides or ketolides. These data suggest that oxygen-independent mechanisms contribute to the bactericidal activity of PMN on these strains of S. aureus. Both live and/or heat-killed bacteria impaired the uptake of telithromycin and roxithromycin (but not that of levofloxacin, a quinolone) in a concentration-dependent manner, owing to a modulation of PMN transductional systems involved in the activation of the macrolide carrier.

Macrolide antibiotics have recently triggered much interest owing to the immunomodulatory potential of some derivatives, particularly in the field of inflammatory diseases. Among the possible mechanisms underlying these anti-inflammatory effects, macrolide-induced inhibition of oxidant production by phagocytes has attracted much attention. We and others have previously reported that erythromycin A-derived macrolides impair the phagocyte oxidative burst, a property linked to the presence of L-cladinose. However, we have also demonstrated that other substituents can be involved in the modulation of phagocyte function. Here we have extended the analysis of structure-activity relationships by studying the effects of five 9-N-alkyl derivatives of erythromycylamine on oxidant production by human neutrophils in vitro. LY211397 (2-methoxyethyl derivative) neither altered cell viability nor superoxide anion production. LY281389 (n-propyl derivative) did not alter cell viability and was slightly more inhibitory than erythromycylamine for the production of superoxide anion; its IC50 (concentration that inhibits 50% of the neutrophil response) was about 18 and 24 microM (versus 72 and 74 pM for erythromycylamine) after 60 min of incubation following fMLP and PMA stimulation, respectively. LY80576 (N-phenyl-3-indolylmethyl derivative), LY281981 (3-phenyl-n-propyl derivative) and LY57843 (benzyl derivative) all displayed cellular toxicity at high pharmacological concentrations after 30 to 60 min of incubation. Interestingly, these latter three drugs exhibited a rapid (5 min incubation) and strong inhibitory effect on the neutrophil oxidative burst from either stimulus, with IC50 values of 3 to 10 pM. Further in-vitro and in-vivo investigations are required to analyze the anti-inflammatory potential of these three derivatives.

We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (V(max), about 2,300 ng/2.5 x 10(6) PMN/5 min; K(m), about 50 microg/ml); (iii) the inhibitory effects of Ni(2+) (a blocker of the Na+-Ca(2+) exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system.

Macrolide antibiotics are strongly concentrated within host cells, a property that sustains their activity against intracellular pathogens and is likely responsible for the modulation of cell metabolism and function. There is extensive literature on the subject of macrolide-induced modulation of immune responses. Erythromycin A derivatives seem to display anti-inflammatory activity in vitro, in some animal models and in various clinical settings such as diffuse panbronchiolitis (DPB). The underlying mechanisms are not yet fully understood: inflammatory cytokine and oxidant production by phagocytes is down-regulated by these drugs, but other possible targets include bacterial virulence factors, bronchial and epithelial cells, etc. Also, a link has been suggested between the macrolide transmembrane carrier system and the P-glycoprotein family, which comprises MDR (multiple drug resistance) and CFTR (cystic fibrosis transmembrane conductance regulator), which are respectively involved in the chemotherapeutic resistance of cancer cells and in the genesis of cystic fibrosis.

Professional phagocytes (polymorphonuclear neutrophils and monocytes/macrophages) are a main component of the immune system. These cells are involved in both host defenses and various pathological settings characterized by excessive inflammation. Accordingly, they are key targets for immunomodulatory drugs, among which antibacterial agents are promising candidates. The basic and historical concepts of immunomodulation will first be briefly reviewed. Phagocyte complexity will then be unravelled (at least in terms of what we know about the origin, subsets, ambivalent roles, functional capacities, and transductional pathways of this cell and how to explore them). The core subject of this review will be the many possible interactions between antibacterial agents and phagocytes, classified according to demonstrated or potential clinical relevance (e.g., neutropenia, intracellular accumulation, and modulation of bacterial virulence). A detailed review of direct in vitro effects will be provided for the various antibacterial drug families, followed by a discussion of the clinical relevance of these effects in two particular settings: immune deficiency and inflammatory diseases. The prophylactic and therapeutic use of immunomodulatory antibiotics will be considered before conclusions are drawn about the emerging (optimistic) vision of future therapeutic prospects to deal with largely unknown new diseases and new pathogens by using new agents, new techniques, and a better understanding of the phagocyte in particular and the immune system in general.

Cytokines, the hallmarks of infectious and inflammatory diseases, modify phagocyte activities and thus may interfere with the immunomodulating properties of antibacterial agents. We have investigated whether various proinflammatory cytokines (interleukin 1 [IL-1], IL-6, IL-8, gamma interferon, tumor necrosis factor alpha [TNF-alpha], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) modify two macrolide properties, i.e., inhibition of oxidant production by polymorphonuclear neutrophils (PMN) and cellular uptake. Roxithromycin and two ketolides, HMR 3647 and HMR 3004, were chosen as the test agents. TNF-alpha and GM-CSF (but not the other cytokines) decreased the inhibitory effect of HMR 3647 only on oxidant production by PMN. Fifty percent inhibitory concentrations were, however, in the same range in control and cytokine-treated cells (about 60 to 70 microgram/ml), suggesting that HMR 3647 acts downstream of the priming effect of cytokines. In contrast, the impairment of oxidant production by roxithromycin and HMR 3004 was unchanged (or increased) in cytokine-treated cells. This result suggests that HMR 3004 (the strongest inhibitory drug, likely owing to its quinoline side chain) and roxithromycin act on a cellular target upstream of cytokine action. In addition, TNF-alpha and GM-CSF significantly (albeit moderately) impaired (by about 20%) the uptake of the three molecules by PMN. The inhibitory effect of these two cytokines seems to be related to activation of the p38 mitogen-activated protein kinase. Our data also illuminate the mechanism underlying macrolide uptake: protein kinase A- and tyrosine kinase-dependent phosphorylation seems to be necessary for optimal uptake, while protein kinase C activation impairs it. The relevance of our data to the clinical setting requires further investigations, owing to the complexity of the cytokine cascade during infection and inflammation.

The mechanism of radiolabeled levofloxacin ([3H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis: r = 0.92; P < 0.001). The activation energy was low (36 +/- 7.2 kJ/mol). Levofloxacin uptake was increased in Ca(2+)-depleted, EGTA-containing medium by approximately 33% (P = 0.022), while Ni2+, a Ca2+ channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an L-type Ca2+ channel inhibitor) and other pharmacologic agents which modify Ca2+ homeostasis did not modify levofloxacin uptake. Interestingly, Ca2+ and Mg2+ inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni2+, and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca2+ pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca2+ and Mg2+ may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake.

Various reviews have highlighted the potential immuno-modulating properties of macrolides. Recent data in this field raise the possibility of new therapeutic prospects in cancer and inflammatory diseases (cystic fibrosis, asthma, atherosclerosis, etc.). Advances have also been made in our understanding of the interactions between macrolides and host immune effectors, particularly phagocytes. The third millennium should see exciting new uses of macrolides.

HMR 3647, a new ketolide, is active upon intracellular pathogens. We previously demonstrated that HMR 3004 (RU 64004), another ketolide, is highly concentrated by human polymorphonuclear neutrophils (PMNs). This prompted us to evaluate whether the presence of a 3-keto group instead of an L-cladinose, a neutral sugar characteristic of erythromycin A derivatives, confers peculiar pharmacokinetic properties with regard to cellular accumulation and efflux. After incubation with the radiolabelled drug, HMR 3647 uptake was determined by a velocity gradient centrifugation technique. HMR 3647 was avidly concentrated by PMNs, without saturation, over a 3-h incubation period, with cellular-to-extracellular concentration ratios of 31 +/- 4.2 at 5 min and up to 348 +/- 27.1 at 180 min. About 60% of HMR 3647 was located in the granular compartment; less than 6% was associated with the membranes. HMR 3647 gradually egressed from loaded cells placed in drug-free medium. Uptake was dependent on environmental temperature (activation energy, 128 +/- 9. 4 kJ/mol) but not on extracellular pH. HMR 3647 displayed Michaelis-Menten saturation kinetics with a mean Vmax of 2315 ng/2.5 x 10(6) PMNs/5 min and a mean Km of 117 mg/liter (144 microM). As already observed with erythromycin A-derived macrolides, extracellular Ca2+ was necessary for optimal uptake of HMR 3647. Interestingly, verapamil increased the uptake of HMR 3647 at 5 min, but this was followed by gradual inhibition at later incubation times, a phenomenon probably related to stimulation of drug efflux. The impact of intracellular accumulation of HMR 3647 on PMN functions was also investigated. In contrast to other erythromycin A derivatives, HMR 3647 only weakly triggered granule exocytosis, but it inhibited superoxide anion production in a time- and concentration-dependent manner, with concentrations which inhibited 50% of control response of 55 (67 microM) (5 min) and 30 (36 microM) (30 min) mg/liter for formyl-methionyl-leucyl-phenylalanine stimulation and 117 (143 microM) (5 min) and 44 (54 microM) (30 min) mg/liter for phorbol myristate acetate stimulation.