JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase.

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Differential Induction Pattern Towards Classically Activated Macrophages in Response to an Immunomodulatory Extract from Pleurotus ostreatus Mycelium.

Pleurotus ostreatus mushroom preparations have been investigated because of their ability to modulate the immune function. However, there is still no consensus regarding the activation and polarizing effect on macrophages by Pleurotus-derived bioproducts. This study examined the immune-activating effect of a mycelium-derived P. ostreatus aqueous extract (HW-Pm) on macrophage functions, by means of the determination of nitric oxide (NO) production, the mRNA expression of inducible nitric oxide synthase (iNOS), Arginase-1 and FIZZ and the cytokine levels. The phagocytic activity and the activation of NF-κB in U937 reporter cells were also investigated. No cytotoxicity was observed in macrophages treated with HW-Pm (IC50 > 1024 µg/mL) by the resazurin test. HW-Pm induced high levels of NO production and iNOS expression in macrophages. In contrast, HW-Pm did not induce Arginase-1 and FIZZ mRNA expressions. The mushroom extract increased TNF-α and IL-6 production and the phagocytic function in murine macrophages. It also stimulated the activation of the NF-κB promoter. The P. ostreatus mycelium extract has a potential application as a natural immune-enhancing agent, by targeting macrophage activation towards the classically activated subset and stimulating macrophage-mediated innate immune responses.

A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

A small-molecule fusion inhibitor of influenza virus is orally active in mice.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

Characterizing the in vitro biofilm phenotype of Staphylococcus epidermidis isolates from central venous catheters.

Central venous catheter (CVC)-related infections are commonly caused by Staphylococcus epidermidis that is able to form a biofilm on the catheter surface. Many studies involving biofilm formation by Staphylococcus have been published each adopting an own in vitro model. Since the capacity to form a biofilm depends on multiple environmental factors, direct comparison of results obtained in different studies remains challenging. This study characterized the phenotype (strong versus weak biofilm-producers) of S. epidermidis from CVCs in four different in vitro biofilm models, covering differences in material type (glass versus polymer) and nutrient presentation (static versus continuous flow). A good correlation in phenotype was obtained between glass and polymeric surfaces independent of nutrient flow, with 85% correspondence under static growth conditions and 80% under dynamic conditions. A 80% correspondence between static and dynamic conditions on polymeric surfaces could be demonstrated as well. Incubation time had a significant influence on the biofilm phenotype with only 55% correspondence between the dynamic models at different incubation times (48h versus 17h). Screening for the presence of biofilm-related genes only revealed that ica A was correlated with biofilm formation under static but not under dynamic conditions. In conclusion, this study highlights that a high level of standardization is necessary to interpret and compare results of different in vitro biofilm models.

Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent.

BACKGROUND: Although epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation. METHODS: Wild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures. RESULTS: In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice. CONCLUSION: We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.

Optimization and validation of an existing, surgical and robust dry eye rat model for the evaluation of therapeutic compounds.

The aim of this research was to optimize and validate an animal model for dry eye, adopting clinically relevant evaluation parameters. Dry eye was induced in female Wistar rats by surgical removal of the exorbital lacrimal gland. The clinical manifestations of dry eye were evaluated by tear volume measurements, corneal fluorescein staining, cytokine measurements in tear fluid, MMP-9 mRNA expression and CD3(+) cell infiltration in the conjunctiva. The animal model was validated by treatment with Restasis(®) (4 weeks) and commercial dexamethasone eye drops (2 weeks). Removal of the exorbital lacrimal gland resulted in 50% decrease in tear volume and a gradual increase in corneal fluorescein staining. Elevated levels of TNF-α and IL-1α have been registered in tear fluid together with an increase in CD3(+) cells in the palpebral conjunctiva when compared to control animals. Additionally, an increase in MMP-9 mRNA expression was recorded in conjunctival tissue. Reference treatment with Restasis(®) and dexamethasone eye drops had a positive effect on all evaluation parameters, except on tear volume. This rat dry eye model was validated extensively and judged appropriate for the evaluation of novel compounds and therapeutic preparations for dry eye disease.

Evaluation of topical antifungal products in an in vitro onychomycosis model.

Many topical commercial products are currently available for the treatment of onychomycosis. However, limited data are available concerning their antifungal activity. Using an in vitro onychomycosis model, the daily application of seven nail formulations was compared to the antifungal reference drug amorolfine (Loceryl(®) ) and evaluated for inhibitory activity against Trichophyton mentagrophytes using an agar diffusion test. Of all commercial nail formulations, only Excilor(®) and Nailner(®) demonstrated inhibitory activity, which was much lower compared to the daily application of Loceryl(®) . However, Excilor(®) showed similar efficacy compared to the conventional weekly application of Loceryl(®) . These results suggest a role for organic acids in the antifungal effect of Excilor(®) (acetic acid, ethyl lactate) and Nailner(®) (lactic acid, citric acid, ethyl lactate) as all tested formulations without organic acids were inactive.

A flow cytometric approach to quantify biofilms.

Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

Development of a novel in vitro onychomycosis model for the evaluation of topical antifungal activity.

A novel in vitro onychomycosis model was developed to easily predict the topical activity potential of novel antifungal drugs. The model encompasses drug activity and diffusion through bovine hoof slices in a single experimental set-up. Results correspond well with the antifungal susceptibility assay and Franz cell diffusion test.

Artemisinins, new miconazole potentiators resulting in increased activity against Candida albicans biofilms.

Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.

Importance of biofilm formation and dipeptidyl peptidase IV for the pathogenicity of clinical Porphyromonas gingivalis isolates.

The ability of Porphyromonas gingivalis to cause adult periodontitis is determined by its arsenal of virulence factors. Here, we investigated the importance of biofilm formation and bacterial dipeptidyl peptidase IV (DPPIV) for the pathogenicity of clinical P. gingivalis isolates. In our study, the isolates with biofilm-forming capacity also showed high DPPIV activity in vitro. Moreover, DPPIV activity increased in P. gingivalis biofilms compared to planktonic cells. In a murine subcutaneous abscess model, the biofilm-forming isolates with high DPPIV activity proved to be pathogenic, while the nonbiofilm formers with low DPPIV activity did not induce abscesses. The biofilm-forming ATCC 33277 strain with low DPPIV activity was not pathogenic in mice either. Our results suggest that biofilm formation and DPPIV activity contribute to the pathogenic potential of P. gingivalis. Furthermore, we show that biofilm formation may enhance P. gingivalis virulence through an increased DPPIV activity. Because of their importance for bacterial colonization and growth, biofilm formation and DPPIV activity could present interesting therapeutic targets to tackle periodontitis.

Short cigarette smoke exposure facilitates sensitisation and asthma development in mice.

Epidemiological studies indicate that cigarette smoke exposure is a risk factor for increased sensitisation and asthma development. The aim of this study was to examine the impact of cigarette smoke on sensitisation and allergic airway inflammation in response to a low dose of house dust mite (HDM), and to obtain potential mechanistic insights. Mice were exposed to low doses of HDM extract combined with air or cigarette smoke exposure, either during allergen sensitisation or during the development of allergic airway disease. Mice concomitantly exposed to low-dose HDM, combined with cigarette smoke for 3 weeks, demonstrated an asthmatic phenotype with significantly increased airway eosinophilia, goblet cell metaplasia, airway hyperresponsiveness and a rise in HDM-specific serum immunoglobulin G1, compared to sole HDM or cigarette smoke exposure. In addition, short cigarette smoke inhalation, during the initial contact with HDM allergens, was sufficient to facilitate sensitisation and development of a complete asthmatic phenotype after rechallenge with HDM. Mechanistically, short cigarette smoke exposure amplified dendritic cell-mediated transport of fluorescein isothiocyanate-labelled HDM allergens to the intrathoracic lymph nodes and generated a local T-helper cell type 2 response. Short cigarette smoke exposure is sufficient to facilitate allergic sensitisation and the development of low-dose HDM-induced allergic asthma, possibly by affecting dendritic cell function.

Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice.

BACKGROUND: Cigarette smoke (CS) is a major risk factor for the development of COPD. CS exposure is associated with an increased risk of bacterial colonization and respiratory tract infection, because of suppressed antibacterial activities of the immune system and delayed clearance of microbial agents from the lungs. Colonization with Staphylococcus aureus results in release of virulent enterotoxins, with superantigen activity which causes T cell activation. OBJECTIVE: To study the effect of Staphylococcus aureus enterotoxin B (SEB) on CS-induced inflammation, in a mouse model of COPD. METHODS: C57/Bl6 mice were exposed to CS or air for 4 weeks (5 cigarettes/exposure, 4x/day, 5 days/week). Endonasal SEB (10 µg/ml) or saline was concomitantly applied starting from week 3, on alternate days. 24 h after the last CS and SEB exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and lung tissue were collected. RESULTS: Combined exposure to CS and SEB resulted in a raised number of lymphocytes and neutrophils in BAL, as well as increased numbers of CD8+ T lymphocytes and granulocytes in lung tissue, compared to sole CS or SEB exposure. Moreover, concomitant CS/SEB exposure induced both IL-13 mRNA expression in lungs and goblet cell hyperplasia in the airway wall. In addition, combined CS/SEB exposure stimulated the formation of dense, organized aggregates of B- and T- lymphocytes in lungs, as well as significant higher CXCL-13 (protein, mRNA) and CCL19 (mRNA) levels in lungs. CONCLUSIONS: Combined CS and SEB exposure aggravates CS-induced inflammation in mice, suggesting that Staphylococcus aureus could influence the pathogenesis of COPD.

Molecular structure of the Mycobacterium tuberculosis virulence factor, mycolic acid, determines the elicited inflammatory pattern.

Mycolic acids (MAs) occur in the cell wall of Mycobacterium tuberculosis as variable mixtures of different classes and chain lengths. Here, we address the relationship between the structure and its inflammatory function of this virulence factor using single synthetic MA isomers, differing in oxygenation class and cis- versus α-methyl-trans proximal cyclopropane orientation. Analysis of bronchoalveolar inflammation, lung histopathology and alveolar macrophage transcription revealed a strong dependence on these meromycolic chemistries of mouse pulmonary inflammation in response to intratracheal treatments with MAs. Whereas α-MA was inert, oxygenated methoxy- and keto-MA with cis-cyclopropane stereochemistry elicited solid to mild inflammatory responses respectively. In trans-cyclopropane orientation, methoxy-MA partially lost its inflammatory activity and keto-MA exerted anti-inflammatory alternative activation of alveolar macrophages and counteracted cis-methoxy-MA induced airway inflammation. The differential innate immune activities of MAs demonstrated here, dependent on oxygenation class and cis versus α-methyl-trans cyclopropane chemistry, identify a novel means for M. tuberculosis to steer host immune responses during infection.

Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation.

Air pollutant exposure has been linked to a rise in wheezing illnesses. Clinical data highlight that exposure to mainstream tobacco smoke (MS) and environmental tobacco smoke (ETS) as well as exposure to diesel exhaust particles (DEP) could promote allergic sensitization or aggravate symptoms of asthma, suggesting a role for these inhaled pollutants in the pathogenesis of asthma. Mouse models are a valuable tool to study the potential effects of these pollutants in the pathogenesis of asthma, with the opportunity to investigate their impact during processes leading to sensitization, acute inflammation and chronic disease. Mice allow us to perform mechanistic studies and to evaluate the importance of specific cell types in asthma pathogenesis. In this review, the major clinical effects of tobacco smoke and diesel exhaust exposure regarding to asthma development and progression are described. Clinical data are compared with findings from murine models of asthma and inhalable pollutant exposure. Moreover, the potential mechanisms by which both pollutants could aggravate asthma are discussed.

Concomitant inhalation of cigarette smoke and aerosolized protein activates airway dendritic cells and induces allergic airway inflammation in a TLR-independent way.

Cigarette smoking is associated with the development of allergic asthma. In mice, exposure to cigarette smoke sensitizes the airways toward coinhaled OVA, leading to OVA-specific allergic inflammation. Pulmonary dendritic cells (DCs) are professional APCs involved in immunosurveillance and implicated in the induction of allergic responses in lung. We investigated the effects of smoking on some of the key features of pulmonary DC biology, including trafficking dynamics and cellular activation status in different lung compartments. We found that cigarette smoke inhalation greatly amplified DC-mediated transport of inhaled Ags to mediastinal lymph nodes, a finding supported by the up-regulation of CCR7 on airway DCs. Pulmonary plasmacytoid DCs, which have been involved in inhalational tolerance, were reduced in number after smoke exposure. In addition, combined exposure to cigarette smoke and OVA aerosol increased surface expression of MHC class II, CD86, and PDL2 on airway DCs, while ICOSL was strongly down-regulated. Although inhaled endotoxins, which are also present in cigarette smoke, have been shown to act as DC activators and Th2-skewing sensitizers, TLR4-deficient and MyD88 knockout mice did not show impaired eosinophilic airway inflammation after concomitant exposure to cigarette smoke and OVA. From these data, we conclude that cigarette smoke activates the pulmonary DC network in a pattern that favors allergic airway sensitization toward coinhaled inert protein. The TLR independency of this phenomenon suggests that alternative immunological adjuvants are present in cigarette smoke.