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Background The nuclear factor kappa-B (NF-κB) is a major transcription factor responsible for the production of numerous inflammatory mediators, including the tumor necrosis factor (TNFα), which has a lethal association with cancer’s onset. The silver nanoparticles (AgNPs) are widely used in cancer treatment and several other biomedical applications. Objective The study aimed to determine the effects of silver citrate nanoparticles (AgNPs-CIT) on NF-κB activation together with TNFα mRNA/protein expressions in the phorbol myristate acetate (PMA)-stimulated MCF-7 human breast cancer cell-lines. Methods The AgNPs-CIT were synthesized by the reduction method, and the prepared AgNPs-CIT were characterized for their shape, absorption in UV-VIS electromagnetic radiations, size distribution, ζ-potential, and antioxidant activity. The MCF-7 cell-lines were pretreated with AgNPs-CIT and stimulated with PMA. The TNFα mRNA expressions were determined by real-time PCR, whereas the protein production was determined by the ELISA. The NF-κB activity was distinctly observed by highly-specific DNA-based ELISA, and by NF-κB-specific inhibitor, Bay 11–7082. Results The prepared AgNPs-CIT were spherical and have an absorption wavelength range of 381–452 nm wherein the particles size ranged between 19.2±0.1 to 220.77±0.12 nm with the charge range −9.99±0.8 to −34.63±0.1 mV. The prepared AgNPs-CIT showed comparative antioxidant activity at >40% inhibitions level of the DPPH radicals. The AgNPs-CIT were found to be non-toxic to MCF-7 cell-lines and inhibited PMA-induced activation of the NF-κBp65, and also the mRNA/protein expression of TNFα. Conclusion This is the first report that showed AgNPs-CIT inhibited TNFα expression via deactivation of the NF-κB signaling event in stimulated breast cancer cells. The results have important implications for the development of novel therapeutic strategies for the prevention/treatment of cancers and/or inflammatory disorders.

Background Commercial herbal medicines (CHMs) marketed as immune boosters are gaining wide popularity in South Africa, in the absence of control and regulatory guidelines. These commercially packaged and labelled herbal preparations, acquired in various retail outlets, are used without consulting either a conventional health provider or a traditional health practitioner. Although they are indicated for immune-boosting purposes, they might exert many other beneficial and unwanted effects on physiological systems. Platelets are crucial in haemostasis and important for the immunological system. The aim was to investigate the effect of the CHMs used to strengthen the immune system on the activity of human platelets. Methods Six CHMs commonly used as African traditional medicines in Pretoria, South Africa, were tested for their effects on healthy, isolated human platelets, using a bioluminescence method. The tested herbal medicines were Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Ngoma™ Herbal Tonic Immune Booster, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster and serial-diluted standards of each from 10 to 10,000 times. The luminol-enhanced luminescence activity of the platelets was measured after incubation with the herbal medicines and activation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). Results Five herbal medicines, namely Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster exerted comparable weak inhibitory effects on both PMA and fMLP-induced platelets, which were concentration dependent at high doses, and inversely related to concentration at low doses. Intlamba Zifo™, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ exhibited weak, but non-systematic stimulatory effects at low doses, which were not statistically significant. Ngoma™ Herbal Tonic Immune Booster had weak, inhibitory effects at high doses and weak stimulatory effects that were inversely related to concentration at low doses. Conclusion The findings suggest a potential beneficial role of the CHMs in the suppression of platelets’ reactivity and in enhancing the immune system. Caution, however, should be exercised as platelet inhibition and stimulation predispose to the risk of bleeding and thrombosis, respectively.

Operating regime of a single enzymatic cascade such as ubiquitously conserved MAPK building block provides insights into the nature of the sensitivity of the steady-state dose response that maps the upstream kinase and the downstream activated substrate concentrations. Steady-state response can be viewed either at a single-cell level in an ensemble of cells or at population-average level. Four operating regimes, viz., hyperbolic, threshold-hyperbolic, signal transducing and saturated, have been identified using the population-average level dose response curve. However, cell-to-cell variability exists in enzymatic cascades. This variability is captured using reporter based experimentation at ensemble level which permits detection of snapshot(s) of ensemble-level distribution of phosphorylated proteins. As a result, often the corresponding underlying steady-state dose response curve may not be available. We consider the question if the underyling operating regime can be directly inferred from ensemble level snapshot upstream kinase (input) and downstream phosphorylated substrates (response) distributions. In order to address this question, we use mathematical model of a single enzymatic cascade based on quasi-steady state approximation superimposed with an input distribution constrained by single-cell level experimental measurements of the MAPK cascade in Jurkat E6.1 cells stimulated with Phorbol Myristate Acetate (PMA). We prove that, under steady-state conditions, a monotonic relationship between the RIQR(=ratio of the inter-quartile range of the response and input distributions) and Rm(=ratio of medians of the two distributions), both of which are experimental observables, can be used to identify the underlying operating regime. We also show that the identification of the unimodal vs bimodal nature of the response distributions can further lead to identification of the potential parameter range in the planes of Michaelis-Menten constantsK1andK2, the two key parameters that dictate the operating regimes. We implement the proposed method on the stimulus strength dependent steady-state single-cell level pMEK (input) and pERK (response) distributions in Jurkat E6.1 cells treated with PMA. While cells stimulated using low concentrations of PMA are likely to operate in hyperbolic regime, those exposed to higher concentrations may lie in signal-transducing regime.Author’s summarySingle enzymatic cascade, a ubiquitously found key building block in biological signaling networks, exhibits different steady-state behaviour at population (ensemble) level compared to population-averaged response. Detection at ensemble level typically achieved by non-plasmid based reporter permits snapshot fluorescence distribution measurement. We ask if there are signatures of fluorescence distribution of input kinase and response activated protein that can help decipher the underlying dose-response belonging to four distinct operating regimes a single enzymatic cascade exhibits. Based on simultaneously measured snapshot input (pMEK) and response (pERK) protein levels in an ensemble of PMA stimulated immortalized cancer (Jurkat E6.1) cells, we superimpose pMEK data-guided upstream kinase distribution capturing cell-to-cell variability on the steady-state Michaelis-Menten (MM) kinetic model. Following extensive MM constants sampling and simulations, we systematically identify that monotonicity between RIQR, the ratio of the inter-quartile range of response and input distributions, and RM, the median ratio of the two distributions, enables regime identification of the measured pERK distribution. We further show qualitative assessment of the modality of the response distribution can constrain the parameter range within an operating regime. Both RIQRand RMbeing experimental observables makes the proposed method suitable for assessing signal modulation capability of an enzymatic cascade of interest.

Multiplexed single-cell protein secretion analysis provides an in-depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co-profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single-cell secretion analysis platform by sandwiching PDMS stencil in between two antibody-coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5-plexed, 3-fluorescence color single-cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single-cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.

Background We previously reported that phorbol 12-myristate 13-acetate (PMA) treatment can induce the cardiac differentiation of mesenchymal stem cells (MSCs). In the present study, we investigated how PMA induces cardiac differentiation of MSCs, focusing on its effect on the transcription factors responsible for increased cardiac marker gene expression. Methods Human MSCs (hMSCs) were treated with 1 μM PMA for 9 days. The expression of MSC markers and cardiac markers in the PMA-treated hMSC, as well as the nuclear translocation of transcription factors, nuclear factor of activated T cells (NFAT), and myogenic differentiation 1 (MyoD), was examined. Transcriptional activity of NFAT was examined by utilizing a green fluorescent protein (GFP) vector containing NFAT motif of human interleukin-2 promoter. The effect of PMA on the expression of key cell cycle regulators was examined. Results PMA induces the transcriptional activity of NFAT and MyoD, which have been associated with increased expression of cardiac troponin T (cTnT) and myosin heavy chain (MHC), respectively. Our data suggested that protein kinase C (PKC) mediates the effect of PMA on NFAT activation. Furthermore, PMA treatment increased cell-cycle regulator p27kip1 expression, suggesting that PMA triggers the cardiac differentiation program in MSCs by regulating key transcription factors and cell cycle regulators. Conclusions The results of this study demonstrate the importance of NFAT activation during PMA-induced MSC differentiation and help us to better understand the underlying mechanisms of small molecule-mediated MSC differentiation so that we can develop a strategy for synthesizing novel and improved differentiation-inducing small molecules.

When determined in vitro, the SPF of certain commercial sunscreen products can be lower than the SPF indicated on the label. The study of the composition of this type of product enabled us to note that the majority contained substances reputed to have anti-inflammatory properties. This effect is shown by inhibiting the erythema, without protecting the skin, which constitutes a serious public health problem. The anti-inflammatory effects of αbisabolol-, allantoin- and 18-β-glycyrrhetinic acid-based emulsions, as well as commercial sun products containing any one of these molecules, have been tested with phorbol myristate acetate on mice. The effectiveness of these sunscreens products is quantified using two indicators: SPF (sun protection factor) and PF-UVA (protection factor-UVA) by in vitro method. We were thus able to show that certain sun products have an anti-inflammatory effect, which in turn causes the SPF value shown on the product to be overestimated, indeed sometimes by considerably large margins.

The objective of this study was to analyze the un-stimulated and stimulated release of superoxide anion (O2 −) by granulocytes and monocytes in juvenile systemic lupus erythematosus (jSLE). The un-stimulated and phorbol myristate acetate (PMA, 30 nM)-induced O2 −by granulocytes and monocytes were determined in six different times of incubation in patients with 23 jSLE and 28 controls. The analysis compared the jSLE group, which was classified into two subgroups by SLEDAI in one inactive subgroup (score

The purpose of this study was to establish a model of delayed type hypersensitivity (DTH) reaction in the ear skin of large animals such as adult Yucatan pigs, which may aid in evaluating the efficacy of therapeutic modalities of newly developed anti-inflammatory drugs. The pigs were sensitized with oxazolone, re-challenged with the same irritant six days later, and dosed with either vehicle or with cyclosporine A (CsA) before and after challenge. CsA reduced the redness, inhibited the accumulation of ear fluid and inflammatory cells, as well as the release of the inflammatory mediators. Further, CsA inhibited the proliferation of T cells collected from the spleens or PBMCs of CsA-treated pigs when these cells were stimulated in vitro with PMA plus Ionomycin. These results indicate that pig skin can be used to evaluate modalities for the purpose of developing drugs that may be used to treat DTH in humans.

The initial arrest and subsequent rolling of a leukocyte on the vascular endothelium is believed to be facilitated by the extraction of tethers, which are narrow membranous tubes drawn from the leukocyte. Although single tether extraction from neutrophils has been studied thoroughly, the relationship between the tether force (F) and tether-growth velocity (Ut) is still unknown for double tethers drawn from neutrophils. In this study, we have determined this relationship with the micropipette-aspiration technique. As a comparison, tether extraction from CD4+ T-lymphocytes was also studied. The threshold force and effective viscosity for single tether extraction from passive CD4+ T-lymphocytes were found to be 46 pN and 1.55 pN · s/μm, respectively. These values were modulated by stimulation with phorbol myristate acetate (PMA), but not interleukin-8 (IL-8). More importantly, for both types of leukocyte, the threshold force and effective viscosity for double tether extraction are about twice as large as those corresponding to single tether extraction. Neither IL-8 nor PMA stimulation had any effect on this correlation. These results indicate that double tethers are highly localized on cellular surfaces and independent of each other during the rolling process.

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals.

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.

Most subunits of the alphabeta deltaepsilon gammaepsilon zetazeta T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRalpha,beta molecules and invariant CD3gamma,delta,epsilon chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRalpha subunits, the role of calnexin in the survival and assembly of remaining TCR components is unknown. Here we evaluated the expression of TCR proteins in CEM T cells and the calnexin-deficient CEM variant CEM.NK(R). We found that CEM and CEM.NK(R) cells constitutively synthesized all TCR subunits except for TCRalpha and that CD3gamma,delta,epsilon components and CD3-beta complexes were effectively assembled together in both cell types. The stability and folding of core CD3epsilon chains were similar in CEM and CEM.NK(R) cells. Interestingly, TCRalpha synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NK(R) cells and TCRalpha proteins synthesized in CEM.NK(R) cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NK(R) cells in the absence of calnexin synthesis. These results demonstrate that TCR complexes can be expressed in the absence of calnexin and suggest that the role of calnexin in the quality control of TCR assembly is primarily restricted to the stabilization of newly synthesized TCRalpha proteins.

A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory burst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory burst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+]i, the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.

Using an MCLA-dependent chemiluminescence technique, we evaluated superoxide production by neutrophils isolated from 7 patients with Behçet's disease. After stimulation by phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan, neutrophils from the patients produced significantly more superoxide than those from 20 controls. Pretreatment of control neutrophils with serum prominently enhanced superoxide production, and serum from Behçet's disease patients had a significantly greater effect than that from controls. These findings suggest that serum from patients with Behçet disease contains the priming factor(s) that can enhance enhanced superoxide production by neutrophils in response to stimulation.

Polymorphonuclear leukocytes (PMNs) are one of the most important components of the defence mechanisms against bacterial infection. The functions of PMNs are believed to be impaired in patients during the perioperative period. Bactericidal function of PMNs was investigated together with the luminol-dependent chemiluminescence (CL) reaction of whole blood in 23 patients, 12 undergoing open surgery and 11 undergoing endoscopic surgery. Blood samples were collected one day before surgery (day -1) and 2 hours (day 0), 24 hours (day 1) and 7 days (day 7) after surgery. Counts of whole white blood cells (WBCs), PMNs and lymphocytes were not different between the two surgery groups. CL responses in the open surgery group were increased on days 0, 1 and 7. In the endoscopic surgery group, CL response was increased on day 1, but not on day 0 or day 7. These results suggest that the PMN function during the perioperative period was not impaired, but increased just after surgery, mainly due to an increasing number of WBC caused by the surgical intervention.

The respiratory burst reaction has been studied in monocytes from men and women of different age. Phorbol myristate acetate (PMA) was used to stimulate NADPH oxidase. Superoxide anion production was found to be dependent on age and sex (it decreased 45% in men and 70% in women during aging).

The effect of phorbol 12-myristate 13-acetate (PMA) on the expression and shedding of intercellular adhesion molecule-1 (ICAM-1) was investigated on the hematopoietic cell lines K 562 and U 937 using flow cytometry, fluorescence microscopy and ELISA technique. At low concentration of 1 nM, PMA stimulated the expression of ICAM-1 on the cell surface about 4-fold within 24 h, whereas a short-term treatment with 101) nM PMA led to the shedding of 35% of ICAM-1 from the surface of K 562 cells. The release of surface ICAM-1 was found on single cells by fluorescence microscopy to be a uniform process proceeding within 15 min. The shedding of ICAM-1 correlated with elevated levels of sICAM-1 in the supernatants of cultured cells. Also on K 562 cells stimulated by TNF-α, a PMA-induced release of ICAM-1 was observed in addition to the known spontaneous shedding. In contrast to the results with K 562 cells, no PMA-induced shedding of ICAM-1 was found on U 937 cells. This indicates a cell-specific process for K 562 cells. The PMA-mediated release of ICAM-1 from K 562 cells suggests that the shedding process does not only occur in parallel to the surface expression of ICAM-1, but may be controlled by particular mechanisms of down-regulation.

This report describes a method for measuring the respiratory burst in neutrophils, based on intracellular oxidation of the reduced ethidium bromide derivative, hydroethidine. Fluorescence of the resultant product quantitatively determined by how cytofluorometry serves as a measure of the neutrophil ability to generate superoxide radicals. We found that during inflammation some polymorphonuclear leukocytes showed a considerably lower respiratory burst response to phorbol myristate acetate treatment. It was demonstrated that variations in this parameter could be an indicator of the course of inflammation.

Aims Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases such as asthma and atopic dermatitis. Schizandrin has various effects such as anti-asthmatic, anti-cancer and anti-inflammatory effects. However, the effect of schizandrin on the production of TSLP has not been clarified. Thus, we investigated how schizandrin inhibits the production of TSLP in the human mast cell line HMC-1 cells. Main methods We used enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, luciferase assay, and Western blot analysis to investigate the effects of schizandrin. Key findings Schizandrin inhibited the production and mRNA expression of TSLP in HMC-1 cells. The maximal inhibition rate of TSLP production by schizandrin (10 μM) was 68.62 ± 3.47%. Schizandrin inhibited the translocation and luciferase activity of nuclear factor-κB induced by phorbol myristate acetate plus A23187. In the activated HMC-1 cells, the activation of caspase-1 was increased, whereas the activation of caspase-1 was decreased by pretreatment with schizandrin. Significance These results suggest that schizandrin can be used to treat inflammatory and atopic diseases through the inhibition of TSLP.

Incubation of HER 14 cells with phorbol myristate acetate (PMA) decreases epidermal growth factor (EGF)-dependent protein tyrosine phosphorylation, except for a 40-kDa MAP kinase II-like protein, whose tyrosine phosphorylation is further enhanced. The inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation is reversed if cells are pre-incubated with a combination of Na3VO4 and NaF, two known inhibitors of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity of cell homogenate was measured on immunopurified EGF receptor, and was found to be enhanced in PMA-treated cells. These data suggest that the inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation in HER14 cells may be mediated by protein tyrosine phosphatase activity.

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10-fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA-stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA-stimulated respiratory burst. Using 1-butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA-stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA-stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA-stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA-stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2-dioctanoyl-sn-glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA-stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA-stimulated respiratory burst.

The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been identified as one of the main factors responsible for the induction of sperm DNA damage. Spermatozoa are mainly protected against ROS-induced damage by seminal plasma. However, this protective effect disappears once spermatozoa enter the female genital tract. The fallopian tube mucosa may play a protective role against ROS-induced sperm damage. The main objective of this study was to determine whether human tubal explants and tubal fluid exert a protective effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to tubal explants and/or tubal fluid in the presence of phorbol myristate acetate (PMA)-activated polymorphonuclear leucocytes or control medium and sperm DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incubation of spermatozoa with tubal explants did not reduce this damage. However, pre-incubation of spermatozoa with tubal fluid resulted in a statistically significant reduction in sperm DNA fragmentation levels, comparable to those observed in control. In conclusion, tubal fluid appears to protect against activated leucocyte-induced sperm DNA fragmentation, thus preserving the integrity of the paternal genome.

The respiratory burst of polymorphonuclear leukocytes was investigated in 24 children with insulin dependent diabetes mellitus and 24 healthy controls. This oxygen dependent, membrane associated process generates a number of toxic oxygen metabolites which are implicated in the pathogenesis of endothelial damage. The activity of polymorphonuclear leukocytes was studied in terms of luminol amplified chemiluminescence. It was found that the resting luminol amplified chemiluminescence activity of isolated polymorphonuclear leukocytes from diabetic children was significantly higher than that of controls (342,000 +/- 174,000 cpm vs. 165,000 +/- 82,000 cpm, p less than 0.01). The addition of respiratory burst inhibitors caused a significant reduction of basal chemiluminescence (greater than 80%). When the ratio of phorbol myristate acetate stimulated activity to basal activity was calculated and used as an activation index, it was found to be significantly reduced in diabetics relative to controls (4.29 +/- 2.46 vs. 8.34 +/- 3.21, p less than 0.01). These observations suggest that increased release of toxic oxygen metabolites from polymorphonuclear leukocytes in diabetic subjects may play a role in the development of diabetic angiopathies.

In twelve rheumatoid arthritis (RA)patients, receiving only nonsteroid antiinflammatory therapy, Superoxide anion (0 2 − ) generation by polymorphonuclear leucocytes (PMNs) was assessed by Cytochrome C reduction. The 0 2 − production by nonactivated PMNs in RA patients was significantly higher than in healthy controls. The 0 2 − production by PMNs activated by zymosan and phorbol myristate acetate was significantly lower than in controls. PMNs from controls preincubated with rheumatoid sera generated higher 0 2 − levels than the same PMNs incubated in normal sera.

IL-4 modulates tissue factor expression by human B lymphocytes in response to phorbol myristate acetate

Adrenergic drugs are often used for hemodynamic support of cardiac output and vasomotor tone in critically ill patients. Recent evidence shows that the administration of these vasoactive drugs may affect cytokine release and could influence the inflammatory response. However, the mechanism of this immunomodulatory effect remains unknown. The nuclear transcription factor-kappaB (NF-kappaB) regulates the expression of many cytokines and plays a central role in the immune response. Therefore, we examined the effects of various adrenergic drugs (dobutamine, xamoterol, clenbuterol, epinephrine, norepinephrine, and phenylephrine) on the activation of NF-kappaB, on the NF-kappaB-driven reporter gene activity, and on the expression of the NF-kappaB target gene interleukin (IL)-8. In addition, we quantified the amount of the NF-kappaB inhibitors IkappaBalpha and IL-10. Here we report that dobutamine inhibited the activation of NF-kappaB in primary human CD3(+) T lymphocytes. Suppression of NF-kappaB involved the stabilization of its inhibitor, IkappaBalpha. The effect appears to be beta(2)-receptor specific, because beta(1)-adrenergic and alpha-adrenergic substances (i.e., xamoterol, epinephrine, norepinephrine, and phenylephrine) did not affect NF-kappaB activation and because dobutamine-mediated inhibition of NF-kappaB could be prevented by a specific beta(2)-antagonist. Our results demonstrate that dobutamine is a potent and specific inhibitor of NF-kappaB, and they thus provide a possible molecular mechanism for the immunomodulation associated with beta-adrenergic therapy.

The anti-inflammatory effect of low-dose radiotherapy is clinically well described. Nevertheless, until now neither the optimal dose nor the background of tissue reactions have been defined. The current study examines the influence of low radiation doses on neutrophilic granulocyte function, which could be helpful in finding the optimal dose for either stimulation or suppression of anti-inflammatory activity.Lymphoprep density gradient-purified neutrophilic granulocytes of three voluntary, healthy donors were used for all experiments. Granulocytes were incubated 48 h in RPMI 1640 and irradiated with single doses of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, and 12 Gy using a (137)Cs IBL 437L irradiator. Their function was assessed by measuring granulocytic release of reactive oxygen species (ROS) with luminol-enhanced chemiluminescence after stimulation with phorbol myristate acetate (PMA).Relative changes of ROS release (ROS release before stimulation was set to 100%) increased after stimulation with PMA (mean +/- standard deviation [SD]): 0 Gy: 147.6% +/- 60%; 0.5 Gy: 153.6% +/- 70%; 1.0 Gy: 164.9% +/- 63%; 1.5 Gy: 177.8% +/- 66%; 2.0 Gy: 162.5% +/- 57%; 2.5 Gy: 156.2% +/- 60%; 3.0 Gy: 159.2% +/- 60%; 3.5 Gy: 126.9% +/- 55%; 4.0 Gy: 137.9% +/- 71%; 6.0 Gy: 148.3% +/- 65%; 12.0 Gy: 156.1% +/- 52%. The relative ROS release showed a significant increase at 1.5 Gy (p0.001) after PMA stimulation and a significant decrease of ROS release at 3.5 Gy (p0.005) and less markedly at 4.0 Gy (p0.05). 6.0 and 12.0 Gy showed a significant (p0.05) increase again.This ex vivo in vitro study on native human neutrophilic granulocytes shows an increase at 1.5 Gy and a significant decrease of granulocyte function at 3.5 and 4.0 Gy, as it was described for different other phenomena in low-dose radiotherapy. These results may provide a further explanation for the local anti-inflammatory effect of low-dose ionizing irradiation.

Vasoactive intestinal peptide (VIP) primed the respiratory burst of human neutrophils in response to phorbol myristate acetate. Maximal and half-maximal effects were achieved at 10 and 0.5 nM VIP respectively. The absence of plasma membrane receptors to VIP in neutrophils suggests that priming of the respiratory burst should be considered as a side effect of VIP. However, from the above indicated concentration range, the priming of the neutrophil by VIP cannot be considered as a pharmacological effect. The enhancement of the formation of reactive oxygen metabolites by VIP may be important in the pathology of VIP-producing tissues.

Results obtained by measuring human whole blood neutrophil chemiluminescence (CL) using the BioOrbit 1251 cuvette luminometer and the Immunotech LM-01T microtitre plate luminometer are compared in this study. Opsonized zymosan, phorbol myristate acetate, N-formyl–Met–Leu–Phe and calcium ionophore A23187 were used as activators. The CL response of neutrophils to their stimulation with the individual types of activators tested was fully detectable using either type of the luminometers. The kinetic curves of CL activity obtained from both the cuvette and the microtitre plate luminometers had similar characteristics. The only insignificant difference observed when comparing the kinetic curves was in the rates of the CL reactions. The peak CL response of activated neutrophils was reached faster when using the luminometer BioOrbit 1251 than with the luminometer Immunotech LM-01T. A likely reason for this difference is the mode of transporting samples during the measurement, inducing different degrees of agitation. However, although this fact needs to be considered when interpreting results, both types of luminometer can be fully utilized in both research and clinical laboratories. Copyright © 2002 John Wiley & Sons, Ltd.

Human alveolar macrophages (AM) and lung tissue macrophages (LTM) have a distinct localization in the cellular environment. We studied their response to direct contact with activated T lymphocytes in terms of the production of interstitial collagenase (MMP-1), 92-kD gelatinase (MMP-9), and of TIMP-1, one of the counter-regulatory tissue inhibitors of metalloproteinases. Either AM obtained by bronchoalveolar lavage or LTM obtained by mincing and digestion of lung tissue were exposed for 48 h to plasma membranes of T lymphocytes previously activated with phorbol myristate acetate and phytohemagglutinin for 24 h. Membranes of activated T cells strongly induced the production of MMP-1, MMP-9, and TIMP-1 exclusively in LTM but not in AM, whereas membranes from unstimulated T cells failed to induce the release of MMPs. Both populations of mononuclear phagocytes spontaneously released only small amounts of MMPs and TIMP-1. Similar results were obtained when MMP and TIMP-1 expression was analyzed at pretranslational and biosynthetic levels, respectively. Blockade experiments with cytokine antagonists revealed the involvement of T-cell membrane-associated interleukin-1 and tumor necrosis factor-alpha in MMP production by LTM upon contact with T cells. These data suggest that the ability of lung macrophages to produce MMPs after direct contact with activated T cells is related to the difference in phenotype of mononuclear phagocytes and cell localization. In addition, these observations indicate that cell-cell contact represents an important biological mechanism in potentiating the inflammatory response of mononuclear phagocytes in the lungs.

A leukotoxin purified from Enterobacter cloacae culture by saline precipitation, gel chromatography and HPLC was studied as a modulator of reactive oxidant species (ROS) produced by human neutrophils. Chemiluminescence showed that stimulation of ROS was achieved at a low leukotoxin concentration, but ROS production decreased when the toxin was applied at concentrations above 30 microg/mL. Also, the addition of 100 microg toxin/mL significantly reduced the activating effect of phorbol myristate acetate (PMA) and low doses of toxin did not produce an opposite effect toward the stimulation produced by PMA. Normal neutrophils showed a linear correlation between the inverse of ROS production and time, but the kinetic reaction changed when toxins were added to the cells and the ROS formation increased directly with time.

The generation both superoxide and a mixture of reactive oxygen species was recorded in a suspension of human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. While stobadine dose-dependently decreased chemiluminescence, only its highest concentration used reduced significantly superoxide generation. The results suggest that stobadine is a more effective scavenger of free radicals rather than a quencher of Superoxide anion.

High-molecular-weight dextran sulfate (HMDS) inhibits infection of CD4+ lymphocytes by T-cell (T)-tropic human immunodeficiency virus (HIV) isolates, but augments replication of macrophage (M)-tropic isolates in primary human macrophages and phorbol myristate acetate (PMA)-differentiated THP-1 monocytic cells. To address the mechanism responsible for HMDS-mediated increases in HIV replication in macrophages, we analyzed the interaction between HMDS and functional domains of gp120 on the surface of PMA-differentiated THP-1 cells infected with M-tropic HIV isolates. Immunofluorescence staining of the infected cells revealed that HMDS inhibited the binding of monoclonal antibodies (mAbs) directed to the V3 and C4 domains of gp120, but augmented the binding of three neutralizing antibodies directed to the V2 region of gp120. The extent of HMDS-mediated changes within the V2 loop of gp120 was associated with increased virus binding and replication in PMA-differentiated THP-1 cells and primary macrophages. The effect was dependent on expression of the CCR5 receptor and was inhibited by the beta-chemokine RANTES. Results of this study suggest that HMDS-mediated increases in HIV infection in macrophages are associated with conformational changes within the V2 region of gp120 and enhanced interaction between gp120 and the CCR5 coreceptor on the target cell.

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

A study was undertaken to assess the ability of the erythrocyte to protect other tissues against oxidative damage. Radiolabelled (51Cr) human umbilical vein endothelial cells (HUVEC) were incubated with erythrocytes and neutrophils activated with phorbol myristate acetate (PMA). Damage to the endothelial cells was indicated by release of radioactivity into the suspending medium. We found that the co-incubation of HUVEC with an increasing range of erythrocyte concentrations resulted in a dose-dependent reduction in the release of radioactivity. When the ability of superoxide to cross the erythrocyte membrane or the glutathione systems was inhibited, the extent of endothelial cell damage increased. Inhibition of the catalase system did not affect results. It was concluded that the erythrocytes afforded some protection against oxidative damage to the endothelial cells by taking up and deactivating the superoxide ions. This protection depends upon intact erythrocyte antioxidant systems. These data support the hypothesis that erythrocytes can provide antioxidant protection to other tissues in vivo.

Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum , which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPγS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPγS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPγS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP 2 .

Sustained Ca2+ influx follows discharge of intracellularly stored Ca2+ in a variety of cell types previously equilibrated in Ca(2+)-free media, including cultured human mesangial cells. This Ca2+ influx pathway has been referred to as capacitative Ca2+ entry or Ca2+ release-activated Ca2+ influx (iCRAC). This study investigated two cellular mechanisms potentially controlling iCRAC in human mesangial cells, protein kinase C (PKC), a key signalling kinase activated by vasoconstrictors that release Ca2+ from internal stores, and calmodulin, a Ca(2+)-binding protein that may couple Ca2+ release to the putative channel(s). The PKC activator phorbol myristate acetate (PMA) dose-dependently inhibited both Ca2+ influx in resting cells and iCRAC, assessed by microfluorometry in fura-2-loaded monolayers, when added before or after 1 uM angiotensin II (AngII) (Ca2+ influx at 1 mM (Ca2+)e +278 +/- 56%/+80 +/- 8%, at 10 mM + 473 +/- 59%/+250 +/- 24% (Ca2+)e, -/+ PMA, respectively, P0.05). PMA did not affect 5 uM ionomycin-induced iCRAC, possibly because it downregulated Ca2+ release by AngII but not by ionomycin, suggesting a key role of released Ca2+ in triggering subsequent Ca2+ influx. This was confirmed by buffering the (Ca2+)i elevation induced by AngII with intracellularly trapped 1,2-bis-(0-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), which abolished any subsequent iCRAC. Moreover, the calmodulin inhibitors calmidazolium (10 uM), trifluoperazine (0.1 mM), or W-7 (0.1 mM) significantly inhibited AngII- or ionomycin-activated iCRAC (+106 +/- 38/229 +/- 53, +58 +/- 9/195 +/- 29, +161 +/- 38/180 +/- 40% at 1/10 mM (Ca2+)e, all P0.05), but did not affect basal Ca2+ entry, consistent with a direct role of cytoplasmic Ca2+ in the regulation of ion gating. These results indicate that iCRAC is under the control of both PKC and calmodulin, and that the site of regulation is distal to the emptying of Ca2+ stores. iCRAC may represent a key mechanism for the control of Ca(2+)-regulated mesangial functions.

1. We describe a rapid and reliable technique for the assessment of basal nitric oxide release in clinical situations, using peripheral blood polymorphonuclear leucocytes isolated by a single-step density gradient procedure. The assay is based on the quantitative conversion of oxyhaemoglobin to methaemoglobin by nitric oxide. We have further examined the ability of these cells to respond to various stimuli. 2. Basal (unstimulated) nitric oxide release occurred, which was augmented by superoxide dismutase. The mean value for healthy subjects was 283 ±96.7 pmolmin−1 10−6 cells. 3. Both phorbol myristate acetate and N-formyl-methionyl-leucylphenylalanine induced further release of nitric oxide, which was increased by preincubation with lipopolysaccharide, interleukin-6 and interferon-γ. 4. Preincubation of cells with NG-monomethyl-l-arginine or l-canavanine sulphate inhibited nitric oxide production. 5. The procedure provides a valuable tool for monitoring nitric oxide up-regulation in clinical situations.

The molecular mode of action of cyclosporin, three of its non-immunosuppressiveanalogs, N-methyl-l-alanyl cyclosporin, acetyl cyclosporin and cyclosporin S 3 , and of FK506 was studied in primary cultures of human thymocytes. Nuclear factors derived from thymocytes activated with phorbol myristate acetate and concanavalin A were tested for their ability to bind to a synthetic radiolabelled probe corresponding to the NF-AT region (−285 to −255) of the IL-2 gene. Binding was observed, and it was inhibited by CsA (100 ng/ml), while the analogs at ten-fold higher concentrations (100 ng/ml) were only partially inhibitory. CsA in combination with FK506 inhibited binding of nuclear factors at the NF-AT site, and acted in concert.

Preexposure of human monocytes to recombinant human interleukin-4 (IL-4) suppressed the respiratory burst response to a number of different stimuli, including phorbol myristate acetate, zymosan, platelet-activating factor and the chemotactic peptide, f-met-leuphe. Under similar conditions, the respiratory burst of murine macrophages was enhanced by preexposure to recombinant murine IL-4. By conducting our studies on cells from different species under similar conditions we have demonstrated that there is a significant disparity in the effects of IL-4 on human and murine macrophages thus providing an explanation for some apparent inconsistencies in the literature and highlighting the need for caution when extrapolating data across species barriers.

The superoxide anion (O2-) production in polymorphonuclear leukocytes stimulated by phorbol myristate acetate in IDDM and non-insulin dependent diabetes mellitus (NIDDM) was determined by the method of Johnston et al, compared with that of each age matched controls. And the correlation between O2- production and hemoglobin (Hb) A1 and A1c value was investigated. The O2- production in IDDM was 24.4 +/- 7.4 (mean +/- SD, n mol per 4 X 10(5) cells) at 10 min. and 51.4 +/- 8.7 at 30 min., in NIDDM each 31.6 +/- 9.3, 60.2 +/- 14.4, and in controls each 40.5 +/- 4.2, 72.4 +/- 3.1. O2- production in IDDM was significantly lower than that in NIDDM (p less than 0.001 at 10 min. and p less than 0.01 30 min.) and controls (p less than 0.001 at 10 and 30 min.). O2- production at 10 and 30 min. possessed a negative correlation with Hba1 and A1c value (HbA1: p less than 0.01 at 10 min. p less than 0.05 at 30 min., HbA1c: p less than 0.01 at 10 and 30 min.). These findings suggest that impaired O2- production might be one of the factors accounting for depressed bactericidal activity of polymorphonuclear leukocytes in IDDM, and that a protracted hyperglycemia might shed some effect on O2- production.

Low density, or hypodense eosinophils (HE) are found in increased numbers in asthma. Since HE have been reported to have increased inflammatory potential and correlate with the severity of airway obstruction, it has been suggested that this subpopulation of eosinophils may contribute to the asthma process. However, investigations to define the function of HE in asthma have been limited. In this study, pure populations of both HE and normal dense eosinophils were isolated from the peripheral blood of seven patients with asthma, and functional activity of these cells was determined by measuring superoxide (O2-) generation to several activators. Compared to normal dense eosinophils, HE generated significantly more O2- when activated with the chemotactic peptide FMLP or opsonized zymosan; however, these differences were small. In contrast, no difference was observed in O2- production between normal- and low-density eosinophils from asthma patients when stimulated by phorbol myristate acetate (PMA) or the calcium ionophore A23187. Furthermore, both eosinophil populations, and corresponding neutrophil isolates, from asthma patients generated significantly more O2- than control granulocytes when activated by PMA. When stimulated by calcium ionophore, all three eosinophil populations released equivalent amounts of O2-, which were, however, higher than from both neutrophil suspensions. Compared to increased activity found in some other hypereosinophilic conditions, blood HE in asthma do not necessarily have enhanced function. This suggests that eosinophil heterogeneity extends not only to cell density but is also reflected in cell function and that these cell variations are dependent on many factors, including the function assessed, source of cells (normal versus patients with some diseases), and stimulus used.

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.

1. 1. The effect of ampicillin on cytochrome c reduction and on the superoxide production of human neutrophils stimulated by phorbol myristate acetate (PMA) was investigated. 2. 2. Ampicillin did not stimulate the superoxide production of intact (resting) neutrophils and not amplify the superoxide production of neutrophils stimulated by phorbol myristate acetate (PMA). 3. 3. However, ampicillin dose-dependently increased the reduction of cytochrome c . 4. 4. In addition, SO mM ampicillin stimulated a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 ± 0.02 (mean ± SD) nmol/min and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 ± 0.03 (mean ± SD) nmol/min. 5. 5. These results suggest that ampicillin serves as an electron donor and/or a superoxide generator.

DMSO differentiated U937 cells responded to 10(-6) M LTD4, LTB4 and FMLP with an increase in both InsP formation and [Ca2+]i. FMLP caused a greater rise in InsPs than either LTD4 or LTB4, which were equivalent. LTD4, however, caused a greater increase in [Ca2+]i than LTB4 (4-fold) or FMLP. The FMLP [Ca2+]i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10(-7) M for 3 min). In contrast, the LTD4 [Ca2+]i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca2+]i and InsP responses to LTD4 by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD4 evoked increases in [Ca2+]i.