The NSs protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis.

UNLABELLED: Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCE: Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.

Transcriptional potencies of inhaled glucocorticoids.

Glucocorticoids (GC) are the most effective anti-inflammatory drugs used in asthma. By a process called trans-activation, they increase the transcription of genes involved in either beneficial processes or certain side effects. Through trans-repression, they inhibit the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1), thereby decreasing the expression of many genes encoding inflammatory mediators such as the cytokine RANTES. We have measured the trans-activation and trans-repression potencies of the five currently available inhaled GC using reporter gene assays. The rank order of trans-activation potencies in HeLa cells stably transfected with a GC-inducible luciferase gene was fluticasone propionate > budesonide and triamcinolone acetonide > beclomethasone dipropionate and flunisolide. For all GC except beclomethasone dipropionate, there was a highly significant correlation between their potency to trans-activate in HeLa cells and their capacity to induce the gluconeogenic enzyme tyrosine aminotransferase in hepatoma tissue culture (HTC) cells. The rank order of trans-repression potencies in A549 lung cells transiently transfected with an AP-1- or NF-kappaB-dependent luciferase gene was fluticasone propionate > budesonide > beclomethasone dipropionate, triamcinolone acetonide, and flunisolide. The same rank order was found for inhibition of RANTES release. Thus, determination of trans-repression and trans-activation potencies of GC may help to predict their capacity to produce anti-inflammatory and side effects, respectively.

VEGF levels in asthmatic airways do not correlate with plasma extravasation.

BACKGROUND: Vascular permeability/vascular endothelial growth factor (VEGF) is a multifunctional cytokine which plays a role in chronic inflammation and angiogenesis. Its expression in bronchoalveolar lavage (BAL) has not been determined although VEGF may be relevant to the pathophysiology of asthma in which oedema is an important feature. METHODS: We studied VEGF, albumin and IgA immunoreactive levels in the BAL fluids obtained from 27 chronic stable asthmatics, nine untreated chronic bronchitis patients and 15 control subjects. RESULTS: BAL fluid levels of VEGF and VEGF normalized to IgA were not significantly different in any patient group. Both asthmatic steroid- and non-steroid-treated groups had significantly lower albumin levels in their BAL fluids explaining most of the 179% increased VEGF normalized to albumin ratios in non-steroid treated asthmatics. Moreover, VEGF and albumin markers correlated in control subjects (r = 0.73, P = 0.006) and in chronic bronchitics (r = 0.75, P = 0.03, Spearman test), but not in asthmatics. VEGF was inversely correlated with asthma severity (GINA/NHLBI scores) in non-steroid treated asthmatics (tau = - 0.52, P = 0.009, Kendall test). CONCLUSIONS: Thus, the potential role of VEGF in asthma requires further studies on bronchial biopsies and induced sputum.

Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects.

Airway remodeling is a well-recognized feature in patients with chronic asthma. The accumulation in the submucosa of fibrous proteins that are substrates of matrix metalloproteinases (MMP), and the demonstration of increased levels of MMP-9 in bronchoalveolar lavage fluid, prompted us to determine whether there was an imbalance between MMPs and tissue inhibitors of metalloproteinase (TIMP) in such patients. We investigated the presence of TIMPs and other MMPs. TIMP levels were compared with those of all MMPs and inflammatory cytokines. Adults with stable asthma, either untreated or treated with glucocorticoids (GCs), were enrolled. Healthy nonsmokers served as a control population. MMPs and TIMPs were identified through zymography or immunoblotting. TIMPs, MMPs, and cytokines were measured with enzyme immunoassays. TIMP-1 levels were significantly higher in untreated asthmatic subjects than in GC-treated subjects or controls (p < 0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, and MMP-9 combined. TIMP-2 was undetectable. TIMP-1 levels were correlated with levels of interleukin-6 (p < 0.012) and the number of alveolar macrophages recovered (p < 0.005). This observation has important implications, since an excess of TIMP-1 could lead to airway fibrosis, a hallmark of airway remodelling in patients with chronic asthma.

Rifampicin is not an activator of the glucocorticoid receptor in A549 human alveolar cells.

It has recently been reported that rifampicin activates the glucocorticoid receptor and acts as an immunosuppressive drug. Because rifampicin constitutes an essential part of pulmonary tuberculosis therapy, we have examined whether it triggers glucocorticoid-like effects in alveolar cells. We have used reporter gene assays to measure the trans-activating and trans-repressing capacity of the glucocorticoid receptor after treating A549 human alveolar cells with rifampicin. The data show that rifampicin neither activated transcription from a promoter containing a glucocorticoid response element nor repressed the activity of activator protein 1 and nuclear factor kappaB, which are transcription factors involved in the immune response. In addition, rifampicin was also unable to inhibit the expression of an endogenous gene that contains activator protein 1 and nuclear factor kappaB response elements and encodes the proinflammatory cytokine RANTES (regulated upon activation normal T expressed and secreted protein). Finally, nuclear translocation of the glucocorticoid receptor, which occurs after ligand binding, was not triggered by rifampicin. In contrast, the glucocorticoid dexamethasone scored positive in all corresponding control experiments. In conclusion, rifampicin is not an activator of the glucocorticoid receptor in A549 alveolar cells. Our results support the clinical observation that rifampicin is not an immunosuppressive drug and suggest that the current medical practice concerning this antibiotic should not be changed.

Increased expression of tissue inhibitor of metalloproteinase-1 and loss of correlation with matrix metalloproteinase-9 by macrophages in asthma.

Alveolar macrophages (AMs) can mediate tissue destruction and repair by synthesizing matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) as well as inflammatory cytokines, which regulate their production. Imbalances between these enzymes and inhibitors may contribute to the tissue damage and remodeling seen in inflammatory diseases. In this study, we examined the role of AMs in chronic asthma. We have previously demonstrated an increased production of MMP-9 by AMs in untreated asthmatic patients as compared with healthy subjects, and in asthmatics treated with inhaled corticosteroids and patients with chronic bronchitis. We now report on the expression of TIMP-1, the inhibitor of MMP-9, and compare the levels and the regulation by cytokines of both MMP-9 and TIMP-1. Enzyme and inhibitor were measured using an enzyme immunoassay and immunoprecipitation. TIMP-1 steady-state mRNA levels were measured using the RNase protection assay. AMs from untreated asthmatics were found to produce more TIMP-1 both at protein and mRNA levels than AMs from other groups. The release of TIMP-1 and MMP-9 from individual AMs was significantly correlated in control populations and the molecules mainly complexed to each other, whereas this was not true for untreated asthmatics, indicating an imbalance between MMP-9 and TIMP-1 production. In the latter population, TIMP-1 release was inhibited by an anti-IL-6 antibody and MMP-9 release by anti-TNF-alpha, anti-IL-6, and anti-IL-1/beta antibodies. The imbalance of MMP-9 and TIMP-1 production, via the involvement of different cytokines, suggests that AMs may be involved in the abnormal repair observed in chronic asthma.

Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis.

Asthma and chronic bronchitis are inflammatory diseases with extracellular matrix (ECM) remodeling and collagen deposition. Collagen homeostasis is controlled by metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). We evaluated MMP and TIMP balance in induced sputum of 10 control, 31 untreated asthmatic, and 16 chronic bronchitic subjects. We first performed zymographic analysis to identify the profile of MMPs. Zymography revealed a similar MMPs profile in all populations studied and that MMP-9 was the major enzyme released. We then measured, using enzyme immunoassay, the concentrations of MMP-9 and of its inhibitor TIMP-1 and evaluated whether airflow limitation may be associated with an imbalance between these enzymes. MMP-9 and TIMP-1 concentrations were greater in sputum of patients with asthma and chronic bronchitis than in control subjects. The molar ratio between MMP-9 and TIMP-1 was lower in asthmatics and chronic bronchitics than in control subjects, and positively correlated with FEV1 values. In asthma, MMP-9 levels were significantly correlated with the number of macrophages and neutrophils. This study shows that airway inflammation in asthma and chronic bronchitis is associated with an imbalance between MMP-9 and TIMP-1 which may have a role in the pathogenesis of ECM remodeling and airflow obstruction.

Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics.

In order to determine whether matrix metalloproteinases (MMPs) contribute to inflammation in asthma, we have examined the release of MMPs in bronchoalveolar lavage (BAL) fluids and their production and regulation by alveolar macrophages (AM), in short-term culture. BAL was collected from 38 asthmatic subjects (24 untreated and 14 treated with inhaled corticosteroids), 26 healthy nonsmokers, and 18 patients with chronic bronchitis used as a control group for another inflammation. The profile of MMPs present in BAL fluid and AM supernatant, determined by zymographic analysis, was found to be similar in all populations. The main enzyme released was identified immunologically as MMP-9, a potent collagenolytic and elastolytic enzyme. Its release, measured using enzyme immunoassay, was significantly enhanced in fluids and in AM supernatants from untreated asthmatics compared with those from the other populations. Enhanced MMP-9 levels, in asthma, could not be explained by a different sensitivity of AM to interleukin-4, interferon-gamma, or dexamethasone, compounds that have been shown to inhibit MMP-9. The phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased MMP-9 in AM from healthy control subjects but not in those from untreated asthmatics. Calphostin C and H7, PKC inhibitors, significantly reduced PMA-stimulated MMP-9 release in AM from healthy control subjects and spontaneous MMP-9 release in AM from untreated asthmatics. H8, a PKA inhibitor, was inactive in both populations. These data suggest that the stimulation of MMP-9 release in AM from untreated asthmatic patients occurs, at least partly, via signals activating PKC.

Heterogeneous spontaneous and interleukin-4-induced nitric oxide production by human monocytes.

The generation of nitric oxide by human monocytes has long been a subject of controversy because of the difficulty of rationalizing this production. In this work we evaluated the capacity of human monocytes to produce nitric oxide (NO) as measured by nitrite (NO2-) release. Resting unstimulated monocytes (2 x 10(6) cells/ml) were found to produce significant amounts of NO2- after 8 to 12 days in culture. This production appeared to be highly heterogeneous. Indeed, approximately, 75% of monocytes from the different donors produced up to 10 microM NO2- and were considered low producers; the last 25% produced higher amounts of NO2- (from 10 to 110 microM) and were considered high producers. In any case the spontaneous production of NO2- by monocytes was overcome in the presence of 1 mM N omega-monomethyl-L-arginine (LNMMA). This inhibitory effect was reversed in the presence of an excess of L-arginine (5 mM), indicating that this process is effectively dependent on L-arginine metabolism. Because interleukin-4 (IL-4) is considered an important NO-regulatory cytokine, its regulatory effect on this spontaneous production of NO was also evaluated. In the presence of a defined dose of IL-4 (1 to 100 ng/ml) the spontaneous production of the high-producing population of monocytes was abrogated, whereas IL-4 stimulated the production by the low-producing population of monocytes, which was suppressed in the presence of LNMMA. The present data indicate that NO production by human monocytes is heterogeneous and that IL-4 can be a potent inducer or inhibitor of this production, suggesting a variability in the activation state of these cells.