Environmental Public Health practice: designing and delivering a locally desirable service.

OBJECTIVES: We reviewed environmental public health practice at a local level (roles, responsibilities, interaction with partner agencies) to establish what and how an integrated approach to the service, as found in Cheshire and Merseyside, North West England, should be delivered, if at all, and at what footprint. STUDY DESIGN: Mixed methods approach. METHODS: We triangulated: qualitative interviews with relevant professionals to gain an in-depth understanding of their interest and vision for any health protection input to health risks and outcomes from environmental issues; an electronic questionnaire assessing experience, interest, vision and comfort zones of a wider range of professionals involved in environmental health issues; a half-day workshop to review study findings and agree ways forward. RESULTS: Stakeholders value their local health protection team's input, but environmental public-health knowledge and skills also exist in local authority teams. Regional health protection teams can provide environmental public-health expertise to local partners and agencies. They harness national input and evidence with local frontline professionals practice, enabling locally grounded approaches, integrating science into local contexts, to answer difficult, often incorrigible, problems. CONCLUSIONS: Specialist leadership by experienced Consultants in Health Protection is of value to local authority public health and environmental teams and should be based on a footprint that is appropriate to enhance local relationships without compromising available expert knowledge and skills.

Antimicrobial resistance in neonates with suspected sepsis.

SETTING: Nobel Medical College and Teaching Hospital, Biratnagar, Nepal. OBJECTIVE: To determine the pattern of antimicrobial resistance and hospital exit outcomes in neonates with suspected sepsis in a tertiary neonatal intensive care unit (NICU). DESIGN: This hospital-based cohort study was conducted to follow patients from January to December 2019. All identified cases of suspected sepsis were enlisted from hospital records. RESULTS: Sepsis was suspected in 177 (88%) of the 200 cases admitted in the NICU; 52 (29%) were culture-positive. Pseudomonas was the predominant organism isolated (n = 40; 78%), followed by coagulase negative staphylococcus (n = 12, 23%). Nine (17%) of the 52 isolates were resistant to the Access and Watch group of antibiotics, including some resistance to Reserve group drugs such as imipenem and linezolid. Most treated cases (n = 170, 96%) improved, although 7 (4%) left against medical advice. CONCLUSION: Most of the pathogens were resistant to WHO Access and Watch antibiotics and occasional resistance was observed to Reserve group drugs. Most sepsis was caused by Gram-negative bacilli. Improving turnaround times for antibiotic sensitivity testing using point-of-care testing, and a greater yield of culture-positive results are needed to enhance the management of neonatal sepsis.

LIEU: Hôpital Universitaire Nobel Medical College, Biratnagar, Népal. OBJECTIF: Déterminer le profil de résistance antimicrobienne et les résultats au moment de la sortie d'hôpital chez des nouveau-nés avec suspicion de sepsis dans une unité néonatale de soins intensifs tertiaires (NICU). MÉTHODE: Cette étude de cohorte hospitalière a été réalisée pour suivre les patients de janvier à décembre 2019. Tous les cas identifiés de suspicion de sepsis ont été inclus à partir des dossiers hospitaliers. RÉSULTATS: Une suspicion de sepsis a été observée chez 177 (88%) des 200 cas admis en NICU ; 52 (29%) étaient positifs par culture. Pseudomonas était le micro-organisme prédominant (n = 40 ; 78%), suivi des staphylocoques à coagulase négative (n = 12, 23%). Neuf (17%) des 52 isolats étaient résistants aux groupes d'antibiotiques « dont l'utilisation est essentielle ¼ et « à utiliser sélectivement ¼, avec certaines résistances aux antibiotiques « de réserve ¼ comme l'imipénème et le linézolide. L'état de la plupart des cas traités (n=170, 96%) s'est amélioré, mais sept (4%) nouveau-nés sont sortis contre avis médical. CONCLUSION: La plupart des pathogènes étaient résistants aux antibiotiques des groupes OMS « dont l'utilisation est essentielle ¼ et « à utiliser sélectivement ¼, et quelques résistances ont été observées à certains « antibiotiques de réserve ¼. La plupart des sepsis ont été causés par des bacilles à Gram négatif. L'amélioration des délais d'exécution des tests de sensibilité aux antibiotiques à l'aide de tests au point de service et un meilleur rendement des résultats positifs par culture sont nécessaires pour renforcer la prise en charge du sepsis néonatal.

High antibiotic resistance and mortality with Acinetobacter species in a tertiary hospital, Nepal.

SETTING: Nepal Mediciti Hospital, Bhainsepati, Lalitpur, Nepal. OBJECTIVES: To determine antimicrobial resistance patterns, and the number and proportion of multidrug-resistant (MDR-) and extensively drug-resistant (XDR-) cases among all patients with Acinetobacter isolates between September 2018 and September 2019. DESIGN: This was a hospital laboratory-based, cross-sectional study. RESULTS: Acinetobacter spp. (n = 364) were more common in respiratory (n = 172, 47.3%) and invasive samples such as blood, body fluids (n = 95, 26.1%). Sensitivity to AWaRe (Access, Watch and Reserve) Group antibiotics (tigecycline, polymyxin B, colistin) remained high. MDR (resistance to at least three classes of antimicrobial agents) (n = 110, 30.2%) and XDR (MDR plus carbapenem) (n = 87, 23.9%) isolates were most common in the Watch Group of antibiotics and found in respectively 99 (31.0%) and 78 (24.5%) patients (n = 319). Infected patients were more likely to be aged >40 years (n = 196, 61.4%) or inpatients (n = 191, 59.9%); 76 (23.8%) patients had an unfavourable outcome, including death (n = 59, 18.5%). CONCLUSION: A significant proportion of MDR and XDR isolates was found; nearly one patient in five died. Robust hospital infection prevention and control measures (particularly for respiratory and invasive procedures) and routine surveillance are needed to reduce infections and decrease the mortality rate. Tigecycline, polymyxin B and colistin should be cautiously used only in MDR and XDR cases.

CONTEXTE: Hôpital de Mediciti, Bhainsepati, Lalitpur, Népal. OBJECTIFS: Déterminer les profils de résistance antimicrobienne, le nombre et la proportion de cas multirésistants (MDR) et ultrarésistants (XDR) parmi tous les patients chez qui des isolats d'Acinetobacter ont été identifiés de septembre 2018 à septembre 2019. MÉTHODE: Il s'agissait d'une étude transversale réalisée dans un laboratoire hospitalier. RÉSULTATS: Acinetobacter spp. (n=364) étaient plus fréquentes dans les échantillons respiratoires (n=172, 47,3%) et invasifs comme le sang et les fluides corporels (n=95, 26,1%). La sensibilité aux antibiotiques de la classification AWaRe (« dont l'accessibilité est essentielle ¼, « à utiliser sélectivement ¼, « de réserve ¼) (tigécycline, polymyxine B, colistine) restait élevée. Les isolats MDR (résistance à au moins trois classes d'agents antimicrobiens) (n=110, 30,2%) et XDR (MDR plus carbapénème) (n=87, 23,9%) étaient plus fréquents dans le groupe des « antibiotiques à utiliser sélectivement ¼ ; ils ont été observés chez respectivement 99 (31,0%) et 78 (24,5%) patients (n=319). Les patients infectés étaient plus susceptibles d'être âgés > 40 ans (n=196, 61,4%) ou hospitalisés (n=191, 59,9%). Un résultat défavorable a été observé chez 76 (23,8%) patients, dont des décès (n=59, 18,5%). CONCLUSION: Une proportion significative d'isolats MDR et XDR a été observée ; près de un patient sur cinq est décédé. Des mesures concrètes de prévention et de contrôle des infections à l'hôpital (notamment pour les procédures invasives et respiratoires) et de surveillance de routine sont nécessaires pour réduire les infections et diminuer le taux de mortalité. La tigécycline, la polymyxine B et la colistine doivent être utilisées avec prudence, uniquement en cas de MDR et XDR.

Pseudomonas aeruginosa in Nepali hospitals: poor outcomes amid 10 years of increasing antimicrobial resistance.

OBJECTIVE: To determine antimicrobial resistance patterns and prevalence of multi- (MDR, i.e., resistant to ⩾3 classes of antimicrobial agents) and extensively (XDR, i.e., resistant to ⩾3, susceptible to ⩽2 groups of antibiotics) drug-resistant strains of Pseudomonas aeruginosa. METHODS: This was a cross-sectional study conducted in Nepal Mediciti Hospital, Lalitpur, Nepal, using standard microbiological methods with Kirby Bauer disc diffusion to identify antimicrobial susceptibility. RESULTS: P. aeruginosa (n = 447) were most frequently isolated in respiratory (n = 203, 45.4%) and urinary samples (n = 120, 26.8%). AWaRe Access antibiotics showed 25-30% resistance, Watch antibiotics 30-55%. Susceptibility to AWaRe Reserve antibiotics remains high; however, 32.8% were resistant to aztreonam. Overall, 190 (42.5%) were MDR and 99 (22.1%) XDR (first Nepali report) based on mainly non-respiratory samples. The majority of infected patients were >40 years (n = 229, 63.2%) or inpatients (n = 181, 50.0%); 36 (15.2%) had an unfavourable outcome, including death (n = 25, 10.5%). Our larger study showed a failure of improvement over eight previous studies covering 10 years. CONCLUSION: Antibiotic resistance in P. aeruginosa occurred to all 19 AWaRe group antibiotics tested. Vulnerable patients are at significant risk from such resistant strains, with a high death rate. Sustainable and acceptable antibiotic surveillance and control are urgently needed across Nepal, as antimicrobial resistance has deteriorated over the last decade.

OBJECTIF: Déterminer les profils de résistance antimicrobienne et la prévalence des souches de Pseudomonas aeruginosa multirésistantes (MDR, c.-à-d., résistantes à ⩾3 classes d'agents antimicrobiens) et ultrarésistantes (XDR, c.-à-d., résistantes à ⩾3, susceptibles à ⩽2 familles d'antibiotiques). MÉTHODES: Il s'agissait d'une étude transversale réalisée à l'Hôpital Mediciti, Lalitpur, Népal, à l'aide de méthodes microbiologiques standards avec test de diffusion sur disque de Kirby Bauer pour identifier la sensibilité aux antimicrobiens. RÉSULTATS: P. aeruginosa (n = 447) a été le plus souvent isolé d'échantillons respiratoires (n = 203, 45,4%) et urinaires (n = 120, 26,8%). Le groupe des « antibiotiques dont l'accessibilité est essentielle ¼ selon la classification AWaRe, a été associé à une résistance de 25­30% contre 30-55% pour ceux du groupe « antibiotiques à utiliser sélectivement ¼. La sensibilité aux « antibiotiques de réserve ¼ restait élevée, mais 32,8% des isolats étaient résistants à l'aztréonam. Dans l'ensemble, 190 (42,5%) isolats étaient MDR et 99 (22,1%) XDR (premier rapport népalais) sur la base d'échantillons principalement non respiratoires. La majorité des patients infectés étaient âgés de >40 ans (n = 229 ; 63,2%) ou hospitalisés (n = 181, 50,0%). Une issue défavorable a été rapportée chez 36 patients (15,2%), dont des décès (n = 25 ; 10,5%). Notre vaste étude a montré l'absence d'amélioration sur huit études antérieures qui couvrent 10 années. CONCLUSION: Une résistance de P. aeruginosa aux 19 antibiotiques de la classification AWaRe testés a été observée. Ces souches résistantes font encourir un risque significatif aux patients vulnérables, avec un taux de mortalité élevé. Un contrôle et une surveillance durable et satisfaisante des antibiotiques sont nécessaires au Népal, puisque le taux de résistance antimicrobienne a augmenté au cours des 10 dernières années.

Molecular Epidemiology of Third-Generation-Cephalosporin-Resistant Enterobacteriaceae in Southeast Queensland, Australia.

Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. In addition to routine mass spectrometry and antibiotic sensitivity testing, isolates were examined using rapid (∼40-min) real-time PCR assays targeting the most common extended-spectrum ß-lactamases (ESBLs; blaCTX-M-1 and blaCTX-M-9 groups, plus blaTEM, blaSHV, and an internal 16S rRNA gene control). AmpC CMY ß-lactamase (blaCMY) prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes, and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one ß-lactamase gene, with 144 (50%) harboring blaCTX-M-1 group, 92 (31.9%) harboring blaCTX-M-9 group, 48 (16.7%) harboring blaSHV, 133 (46.2%) harboring blaTEM, and 34 (11.8%) harboring blaCMY genes. A subset of isolates (n = 98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants and to verify genotyping accuracy. WGS of ß-lactamase-negative or carbapenem-resistant isolates identified uncommon ESBL and carbapenemase genes, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

Beyond 'cure' and 'treatment success': quality of life of patients with multidrug-resistant tuberculosis.

SETTING: Two drug-resistant tuberculosis (DR-TB) sites (MSF Clinic, Jupiter Hospital) in Mumbai, India. OBJECTIVE: To assess health-related quality of life (HRQoL) and associated factors among DR-TB patients and explore their perspectives about HRQoL. DESIGN: We used a mixed-methods design: a quantitative cross-sectional questionnaire (the World Health Organization's Quality of Life Brief Questionnaire [WHOQoL-BREF]); and qualitative in-depth interviews for purposively selected patients. Assessments were conducted between April and November 2016. RESULTS: Ninety-five patients completed WHOQoL-BREF; 12 were interviewed. The psychological and physical health domains were the most affected (mean scores 56.2 ± standard deviation [SD] 18.3, and 56.5 ± SD 15.1, respectively; maximum 100). The social relations and environmental domains mean scores were respectively 68.6 (SD ±21.1) and 60.3 (SD ±15.9). Loss of jobs due to TB adversely affected the social relations and environmental domains. Qualitative analysis showed that support was the most important theme affecting quality of life. Other themes were physical factors (e.g., treatment adverse events), psychological factors (e.g., depression), social functioning (e.g., fear of stigmatisation) and environmental factors (e.g., health systems). CONCLUSION: HRQoL was lower among study participants, but not as low as previously reported among TB patients. Support was the main factor that positively affected HRQoL, although both disease and treatment were physically and socially challenging.

On the Role of Imitation on Adolescence Methamphetamine Abuse Dynamics.

Adolescence methamphetamine use is an issue of considerable concern due to its correlation with later delinquency, divorce, unemployment and health problems. Understanding how adolescents initiate methamphetamine abuse is important in developing effective prevention programs. We formulate a mathematical model for the spread of methamphetamine abuse using nonlinear ordinary differential equations. It is assumed that susceptibles are recruited into methamphetamine use through imitation. An epidemic threshold value, [Formula: see text], termed the abuse reproduction number, is proposed and defined herein in the drug-using context. The model is shown to exhibit the phenomenon of backward bifurcation. This means that methamphetamine problems may persist in the population even if [Formula: see text] is less than one. Sensitivity analysis of [Formula: see text] was performed to determine the relative importance of different parameters in methamphetamine abuse initiation. The model is then fitted to data on methamphetamine users less than 20 years old reporting methamphetamine as their primary substance of abuse in the treatment centres of Cape Town and parameter values that give the best fit are chosen. Results show that the proportion of methamphetamine users less than 20 years old reporting methamphetamine as their primary substance of abuse will continue to decrease in Cape Town of South Africa. The results suggest that intervention programs targeted at reducing adolescence methamphetamine abuse, are positively impacting methamphetamine abuse.

Modelling Drug Abuse Epidemics in the Presence of Limited Rehabilitation Capacity.

The abuse of drugs is now an epidemic globally whose control has been mainly through rehabilitation. The demand for drug abuse rehabilitation has not been matched with the available capacity resulting in limited placement of addicts into rehabilitation. In this paper, we model limited rehabilitation through the Hill function incorporated into a system of nonlinear ordinary differential equations. Not every member of the community is equally likely to embark on drug use, risk structure is included to help differentiate those more likely (high risk) to abuse drugs and those less likely (low risk) to abuse drugs. It is shown that the model has multiple equilibria, and using the centre manifold theory, the model exhibits the phenomenon of backward bifurcation whose implications to rehabilitation are discussed. Sensitivity analysis and numerical simulations are performed. The results show that saturation in rehabilitation will in the long run lead to the escalation of drug abuse. This means that limited access to rehabilitation has negative implications in the fight against drug abuse where rehabilitation is the main form of control. This suggests that increased access to rehabilitation is likely to lower the drug abuse epidemic.

Inflammation: maladies, models, mechanisms and molecules.

The continued focus of attention on the diversity of mechanisms underpinning inflammation has improved our understanding of the potential to target specific pathways in the inflammatory network to achieve meaningful therapeutic gains. In this themed issue of the British Journal of Pharmacology our scope was deliberately broad, ranging across both acute and chronic disease in various organs. Pro- and anti-inflammatory mechanisms receive attention as does the phenotype of macrophages. Whilst the manifestations of neuro-inflammation are less obvious than those in peripheral tissues, central innate and adaptive immunity in brain and the M1/M2 phenotypes of microglia are topics of special interest. The contributions to the inflammatory milieu of cytokines, chemokines and associated signalling cascades are considered. Overall, the coverage herein advances the basic science underpinning our understanding of inflammation and emphasizes its importance in different pathologies.

Modelling the trends of inpatient and outpatient rehabilitation for methamphetamine in the Western Cape province of South Africa.

BACKGROUND: Dependence on methamphetamine remains one of the major health and social problem in the Western Cape province of South Africa. We consider a mathematical model that takes into account two forms of rehabilitation, namely; inpatient and outpatient. We examine the trends of these two types of rehabilitation. We also seek to investigate the global dynamics of the developed methamphetamine epidemic model. METHODS: The model is designed by likening the initiation process to an infection that spreads in a community through interactions between methamphetamine users and non-users. We make use of Lyapunov functions obtained from a suitable combination of common quadratic and Volterra-type functions to establish the global stability of the methamphetamine-persistent steady state. The least squares curve fit routine (lsqcurvefit) in Matlab with optimization is used to estimate the parameter values. RESULTS: The model analysis shows that the model has two equilibria, the methamphetamine free equilibrium and the methamphetamine persistent equilibrium, that are both globally stable when the threshold R(a) < 1 and R(a) > 1, respectively. Upon fitting the model to data on drug users under rehabilitation, parameter values that give the best fit were obtained. The projections carried out the long term trends of these forms of rehabilitation. CONCLUSION: The results suggest that inpatient rehabilitation programs have an increased potential of enhancing the chances of recovery for methamphetamine addicts.

Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling.

BACKGROUND AND PURPOSE: The conversion of plasminogen into plasmin by interstitial urokinase plasminogen activator (uPA) is potentially important in asthma pathophysiology. In this study, the effect of uPA-mediated plasminogen activation on airway smooth muscle (ASM) cell proliferation was investigated. EXPERIMENTAL APPROACH: Human ASM cells were incubated with plasminogen (0.5-50 µg·mL(-1) ) or plasmin (0.5-50 mU·mL(-1) ) in the presence of pharmacological inhibitors, including UK122, an inhibitor of uPA. Proliferation was assessed by increases in cell number or MTT reduction after 48 h incubation with plasmin(ogen), and by earlier increases in [(3) H]-thymidine incorporation and cyclin D1 expression. KEY RESULTS: Plasminogen (5 µg·mL(-1) )-stimulated increases in cell proliferation were attenuated by UK122 (10 µM) or by transfection with uPA gene-specific siRNA. Exogenous plasmin (5 mU·mL(-1) ) also stimulated increases in cell proliferation. Inhibition of plasmin-stimulated ERK1/2 or PI3K/Akt signalling attenuated plasmin-stimulated increases in ASM proliferation. Furthermore, pharmacological inhibition of cell signalling mediated by the EGF receptor, a receptor trans-activated by plasmin, also reduced plasmin(ogen)-stimulated cell proliferation. Knock down of annexin A2, which has dual roles in both plasminogen activation and plasmin-signal transduction, also attenuated ASM cell proliferation following incubation with either plasminogen or plasmin. CONCLUSIONS AND IMPLICATIONS: Plasminogen stimulates ASM cell proliferation in a manner mediated by uPA and involving multiple signalling pathways downstream of plasmin. Targeting mediators of plasminogen-evoked ASM responses, such as uPA or annexin A2, may be useful in the treatment of asthma.

Secreted factors from human mast cells trigger inflammatory cytokine production by human airway smooth muscle cells.

BACKGROUND: A notable feature of allergic asthma is the infiltration of mast cells into smooth muscle in the human airway. Thus, mast cells and human airway smooth muscle (hASM) cells are likely to exhibit mutual functional modulation via direct cell-cell contact or through released factors. This study examined mast cell modulation of hASM cell cytokine release. METHODS: The mast cell line HMCα was used to model mast cell function. hASM cells were either co-cultured directly with resting or IgE/antigen-stimulated HMCα cells or treated with HMCα-conditioned media to examine the impact on cytokine release. The activation pathways triggered in hASM cells by the mast cell-derived factors were examined through the use of selective inhibitors and by Western blotting. RESULTS: HMCα cells, or their conditioned media, induced the expression of cytokines (IL-8 and IL-6) by hASM cells at both the mRNA and the protein level. Cytokine expression in hASM cells was greatly amplified when HMCα cells were IgE/antigen-activated. The effects of the conditioned media were not mediated by the chemokines MCP-1 and MIP-1α or by exosomes. While the mast cell-derived factor(s) increased p38(MAPK) phosphorylation in hASM cells, cytokine production was not inhibited by the p38(MAPK) inhibitor SB203580. hASM cell production of IL-8 induced by HMCα condition media but not IL-6 was, however, attenuated by the Src tyrosine kinase inhibitor PP2. CONCLUSIONS: Our study shows that the release of soluble mediators by activated mast cells can stimulate hASM cells to elicit production of proinflammatory cytokines that may then exacerbate airway inflammation in asthma.

Transforming growth factor-ß impairs glucocorticoid activity in the A549 lung adenocarcinoma cell line.

BACKGROUND AND PURPOSE: The lung adenocarcinoma cell line, A549, undergoes epithelial-mesenchymal cell transition (EMT) in response to TGF-ß. Glucocorticoids do not prevent the EMT response, but TGF-ß induced resistance to the cytokine-regulatory action of glucocorticoids. We sought to characterize the impairment of glucocorticoid response in A549 cells. EXPERIMENTAL APPROACH: A549 cells were exposed to TGF-ß for up to 96 h before glucocorticoid treatment and challenge with IL-1α to assess glucocorticoid regulation of IL-6 and CXCL8 production. Nuclear localization of the glucocorticoid receptor α (GRα) was ascertained by immunofluorescence and Western blotting. Transactivation of the glucocorticoid response element (GRE) was measured with a transfected GRE-secreted human placental alkaline phosphatase reporter. KEY RESULTS: TGF-ß (40-400 pM) reduced the maximum inhibitory effect of dexamethasone on IL-1α-induced IL-6 and CXCL8 production. The impaired glucocorticoid response was detected with 4 h of TGF-ß (40 pM) exposure (and 4 h IL-1α to induce CXCL8 expression) and therefore was not secondary to EMT, a process that requires longer incubation periods and higher concentrations of TGF-ß. TGF-ß also impaired dexamethasone regulation of granulocyte-macrophage colony-stimulating factor in thrombin-stimulated BEAS-2B epithelial cells. Impaired regulation of CXCL8 was associated with markedly reduced GRE transactivation and reduced induction of mRNA for IκBα, the glucocorticoid-inducible leucine zipper and the epithelial sodium channel (SCNN1A). The expression, cellular levels and nuclear localization of GRα were reduced by TGF-ß. CONCLUSIONS AND IMPLICATIONS: We have identified mechanisms underlying the impairment of responses to glucocorticoids by TGF-ß in the A549 and BEAS-2B cell lines.

The influence of nitric oxide synthase 2 on cutaneous wound angiogenesis.

BACKGROUND: Inducible nitric oxide synthase (nitric oxide synthase 2, NOS 2) inhibition significantly suppresses chronically ischaemic skin flap survival, possibly because of reduced angiogenesis. OBJECTIVES: To investigate the effect of genetic NOS 2 inhibition on cutaneous wound angiogenesis in two in vivo murine models. The impact of NOS 2 manipulation on vascular endothelial growth factor (VEGF)-A stimulated and fibroblast growth factor (FGF)-2 stimulated angiogenesis was also investigated in the Matrigel(®) plug assay. METHODS: (i) Matrigel plugs/incisional wounds: two groups of NOS 2-/- mice and two groups of wild-type (WT) mice had bilateral Matrigel plugs containing 500 ng mL(-1) VEGF-A or 1000 ng mL(-1) FGF-2 injected subcutaneously in the abdomen. A 2·5 cm long dorsal incisional skin wound was created and sutured closed in the same animals. Wounds and plugs were explored at 7 or 12 days. (ii) Excisional wounds: dorsal 0·5 × 1·0 cm excisional skin wounds were created in four groups (two NOS 2-/- and two WT) and explored at 7 or 14 days. Wounds and Matrigel plugs were examined histologically and morphometrically for determination of percentage vascular volume (PVV). RESULTS: The PVV in NOS 2-/- incisional wounds and excisional wounds was significantly less than in WT wounds (P = 0·05 and P < 0·001, respectively). The PVV was significantly less in VEGF-A stimulated Matrigel plugs compared with FGF-2 stimulated plugs in NOS 2-/- mice (P < 0·01), but not in WT mice. CONCLUSIONS: NOS 2 is significantly involved in angiogenic signalling in healing skin wounds, particularly within the first 7 days. However, Matrigel plug vascularization suggests that the role of NOS 2 in angiogenesis is related to VEGF-A but not FGF-2 stimulated angiogenesis.

In vitro and in vivo evidence for anti-inflammatory properties of 2-methoxyestradiol.

2-Methoxyestradiol (2MEO) is an endogenous metabolite of 17ß-estradiol that interacts with estrogen receptors and microtubules. It has acute anti-inflammatory activity in animal models that is not attributable to known antiproliferative or antiangiogenic actions. Because macrophages are central to the innate inflammatory response, we examined whether suppression of macrophage activation by 2MEO could account for some of its anti-inflammatory effects. Inflammatory mediator production stimulated by lipopolysaccharide (LPS) and interferon-γ in the J774 murine macrophage cell line or human monocytes was measured after treatment with 2MEO or the anti-inflammatory agent dexamethasone. The effect of these agents on LPS-induced acute lung inflammation in mice was also examined. 2MEO suppressed J774 macrophage interleukin-6 and prostaglandin E2 production (by 30 and 47%, respectively, at 10 µM) and human monocyte tumor necrosis factor-α production (by 60% at 3 µM). Estradiol had no effect on J774 macrophage activation, nor did the estrogen receptor antagonist 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17ß-diol (ICI 182,780) prevent the effects of 2MEO. The actions of 2MEO were not mimicked by the microtubule-interfering agents colchicine or paclitaxel. In mice exposed to LPS, bronchoalveolar lavage protein content, a measure of vascular leak and epithelial injury, was reduced to a comparable extent (~54%) by treatment with 2MEO (150 mg · kg⁻¹) or dexamethasone (1 mg · kg⁻¹). In addition, 2MEO reduced LPS-induced interleukin-6 gene expression. Thus, 2MEO modulates macrophage activation in vitro and has high-dose acute anti-inflammatory activity in vivo. These findings are consistent with the acute anti-inflammatory actions of 2MEO being mediated in part by the suppression of macrophage activation.

Collagen remodelling by airway smooth muscle is resistant to steroids and ß2-agonists.

Bi-directional interactions between airway smooth muscle (ASM) and the altered extracellular matrix (ECM) may influence airway wall remodelling and ASM function in asthma. We have investigated the capacity of cultured human ASM to reorganise the structure of three-dimensional collagen gels and the effects of endothelin (ET)-1 and agents used to treat asthma. Human ASM cells were cast in type I collagen gels. Reductions in gel area over 72 h were determined in the absence and presence of ET-1 and potential inhibitors, steroids and ß2-adrenoceptor agonists. Changes in gel wet weights and hydroxyproline content were measured and ASM gel morphology was examined by scanning electron microscopy. Cell density-dependent reductions in gel area were augmented by ET-1, mediated via ET(A) receptors. This process was not associated with ASM contraction or proliferation, but was consistent with ASM tractional remodelling and migration leading to collagen condensation rather than collagen degradation within gels. The collagen remodelling by ASM was unaffected by salbutamol and/or budesonide. This study demonstrates an additional potential role for ASM in ECM regulation and dysregulation in airways disease that is resistant to steroids and ß2-adrenoceptor agonists. Therapy-resistant collagen condensation within ASM bundles may facilitate ECM-ASM interactions and contribute to increased internal airways resistance.

Mediators and receptors in the resolution of inflammation: drug targeting opportunities.

The active resolution of inflammation is recognized as offering new opportunities to generate novel anti-inflammatory agents. The emerging appreciation of the importance of active resolution in regulation of inflammation also creates an imperative to examine developing and existing agents for their potential to influence these pathways. This themed issue of the British Journal of Pharmacology contains papers that discuss the roles of annexin-1, lipoxins and related lipid products of fish oils as well as other mechanisms involved in active resolution and their receptor targets.

Proliferation is not increased in airway myofibroblasts isolated from asthmatics.

Airway mesenchymal cells, such as myofibroblasts and airway smooth muscle cells, contribute to inflammation, airway remodelling and hyperresponsiveness in asthma by excessive proliferation and inflammatory mediator production. Using endobronchial biopsies obtained from both nonasthmatic and asthmatic subjects, in situ proliferation was assessed by immunostaining for cyclin D1. The number of immunoreactive cells increased with asthma severity and was restricted to the epithelium and subepithelial connective tissue. Despite increases in smooth muscle area, cyclin D1 was not detected in cells in intact muscle bundles. Biopsy-derived cell cultures were characterised as predominantly myofibroblasts, and were assessed to determine whether proliferation and cytokine production varied with asthma status. Cell enumeration showed that basal proliferation was similar in cells from nonasthmatics and asthmatics, and mitogenic responses to fibroblast growth factor-2, thrombin or serum were either reduced or unchanged in cells from asthmatics. Interleukin (IL)-1-dependent granulocyte-macrophage colony-stimulating factor and IL-8 release was increased in cell supernatants from asthmatics. Thus, increased rates of cellular proliferation identified in situ in the asthmatic airway occurred outside the expanded smooth muscle compartment. Although reduced proliferative responses were observed in cultured myofibroblasts from asthmatics, the increased cytokine production by these cells suggests that this contributes to and may perpetuate ongoing inflammation in asthma.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling.

BACKGROUND AND PURPOSE: Airway wall remodelling in asthma is characterised by a number of structural changes, including an increase in the volume of airway smooth muscle (ASM), and the abundance of the extracellular matrix (ECM) protein, collagen, is increased. We have investigated the mechanism of collagen-induced glucocorticoid resistance of proliferation, and migration of ASM. EXPERIMENTAL APPROACH: ASM cultured from human airways has been seeded on to either type I monomeric collagen or a laminin pentapeptide, YIGSR. The role of alpha2beta1 integrin in the collagen-induced glucocorticoid resistance was investigated using a function blocking monoclonal antibody. KEY RESULTS: Culture of ASM on collagen I, but not laminin, led to a greater proliferative response that was insensitive to regulation by dexamethasone (100 nM). The anti-migratory effects of the glucocorticoid, fluticasone propionate (1 nM) were also impaired by contact of ASM with collagen. The impaired anti-mitogenic action of dexamethasone was associated with a failure to reduce the levels of the rate-limiting cell cycle regulatory protein, cyclin D1. When signalling through the alpha2beta1 integrin was reduced, dexamethasone-mediated reductions in proliferation and cyclin D1 levels were restored. CONCLUSIONS AND IMPLICATIONS: In the collagen-rich microenvironment of the inflamed and fibrotic asthmatic airway, integrin/ECM interactions may contribute to glucocorticoid resistance.