The in vitro efficacy of neutral electrolysed water and povidone-iodine against CRS-associated biofilms.

BACKGROUND: Despite best medical and surgical practice, some cases of chronic rhinosinusitis (CRS) can remain recalcitrant. Bacterial biofilms have been associated with the recalcitrance of sinonasal inflammation. Biofilms are highly resistant to commonly prescribed antibiotics. Accordingly, more effective antimicrobial treatment options are needed to treat refractory CRS. The aim of this study was to determine the in vitro efficacy of neutral electrolysed water (NEW) and povidone-iodine (PVI) against CRS-associated Staphylococcus aureus biofilms. METHODS: Mature S. aureus biofilms were grown in a Centre for Disease Control (CDC) biofilm reactor. The antimicrobial activity of NEW, PVI and doxycycline was determined for both planktonic and biofilm cultures of a clinical S. aureus isolate using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) assays. RESULTS: MICs and MBCs were determined for all antimicrobials. MBC values were similar to MICs for both antiseptics, but doxycycline MBCs were significantly higher than the associated MICs. Biofilms were highly resistant to NEW and doxycycline. The MBEC for doxycycline was between 500 and 1000 Â#181;g/mL. NEW was ineffective against biofilms and no MBEC could be determined. In contrast, a concentration of 10% of the commercial PVI solution (10 mg/mL PVI) led to effective eradication of mature biofilms. CONCLUSION: In this study, only PVI showed promising antibiofilm activity at physiological concentrations. The in vivo efficacy of PVI warrants further investigation of its potential as a treatment for recalcitrant CRS.

The sinonasal mycobiota in chronic rhinosinusitis and control patients.

BACKGROUND: While bacterial associations with chronic rhinosinusitis (CRS) are increasingly well described, fewer studies have examined the fungal component of the sinonasal microbiota. Here we present a study of the sinonasal mycobiota in a cohort of 144 patients (106 patients with CRS and 38 controls). METHODOLOGY: Fungal communities were characterised by analysis of mucosal swab samples of the left and right middle meatuses via ITS2 marker amplicon sequencing on the Illumina MiSeq platform. Fungal associations with previously published bacterial community and inflammatory cytokine and cell data for this cohort (collected at the same intra-operative time point) were also investigated. RESULTS: Malassezia spp. were ubiquitous and often highly predominant. Season of sampling explained more of the variability in the data than any of the clinical parameters. The predominant Malassezia sp. was distinct in patients with cystic fibrosis compared to those without. However, distinctions in the mycobiota were not evident between any other patient groupings assessed, and few fungal-bacterial or fungal-inflammatory associations were observed. CONCLUSIONS: This study confirms the prominent place of Malassezia spp. within the upper respiratory tract. Overall, few distinctions between patient groups were evident, and these data lend further support to the hypothesis that fungal community types may have no direct causative association with idiopathic CRS. Additional studies incorporating a broader array of inflammatory markers are required to assess whether these ubiquitous fungi nonetheless play an exacerbating role in some sensitive individuals.

Moving beyond descriptions of diversity: clinical and research implications of bacterial imbalance in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a debilitating disease which affects 5-16% of the general population and involves long-term inflammation of the sinonasal cavity. While microbial involvement in the pathogenesis of CRS has long been suspected, the exact role of microbes remains unclear. Recent application of cultivation-independent, molecular methods has provided much new information, taking advantage of developments in both laboratory- and bioinformatics-based analyses. The aim of this mini-review is to present a variety of available bioinformatics approaches, such as data classification techniques and network analyses, with proven applications in other aspects of human microbiome health and disease research. The uses of molecular techniques in the clinical setting are still in its infancy, but these tools can further our understanding of microbial imbalance during chronic disease and help guide effective patient treatment. The mini-review emphasises ways in which CRS bacterial gene-targeted sequencing data can progress beyond descriptive summaries and toward unlocking the mechanisms by which bacterial communities can be markers for sinus health.

Toll-like receptor activation by sino-nasal mucus in chronic rhinosinusitis.

BACKGROUND: The sino-nasal disease chronic rhinosinusitis (CRS) is primarily an inflammatory condition that manifests in several ways. However, the aetiology of this complex disease is poorly understood. The aim of this study was to explore the association between toll-like receptor (TLR) activation, host immune response and sino-nasal mucus in healthy and diseased patients. METHODS: The activation of TLR2/1 and TLR4 by sino-nasal mucus from 26 CRS patients and 10 healthy controls was measured. In addition, 7 inflammatory cytokines, bacterial community composition and bacterial abundance within the sino-nasal mucus were measured using molecular and diagnostic tools. RESULTS: TLR activity was observed in 9/36 samples, including 2 healthy controls. There was a strong, positive correlation between members of the Gammaproteobacteria (Haemophilus, Enterobacter, Pseudomonas) and TLR2/1 and TLR4 activity. Bacterial abundance and cytokine (tumour necrosis factor) abundance were also positively correlated with TLR activity. CONCLUSIONS: These findings suggest that a small proportion (20-30%) of individuals in each sub-group are more predisposed to TLR activity, which may be related to bacterial composition, diversity and abundance in the sinuses.

Introduction of a new incentive and target-based contract for family physicians in the UK: good for older patients with diabetes but less good for women?

AIMS: To determine whether the recording of diabetes-related health indicators has increased and differences diminished between age, gender and deprivation groups, following the introduction of the new General Medical Services contract (nGMS), an incentive- and target-based contract for UK family physicians. METHODS: A serial cross-sectional study set in 310 primary care practices in Scotland serving a population of 1.5 million registered patients, focussing on diabetic patients. Data were taken immediately before the introduction of the nGMS and after it had been in place for 1 year. RESULTS: One year after the introduction of the nGMS contract, there was a 54.2% relative increase in the number of patients electronically recorded as having diabetes. In addition, measurement of the quality indicators glycated haemoglobin (HbA(1c)), blood pressure, serum creatinine and cholesterol significantly increased (P < 0.05). Women were less likely than men to have HbA(1c)[odds ratio (OR) 0.85, 95% confidence intervals (CI) 0.80-0.91], serum creatinine (OR 0.90, 95% CI 0.84-0.96) and cholesterol recorded (OR 0.83, 95% CI 0.77-0.90) or achieve HbA(1c) (

Gender, age and deprivation differences in the primary care management of hypertension in Scotland: a cross-sectional database study.

BACKGROUND: Little is known about the community management of cardiovascular disease among different gender, age or deprivation groups, even though much of the long-term treatment takes place within primary care. OBJECTIVES: Our aim was to determine whether important gender, age and deprivation differences exist in the primary care management of hypertension. METHODS: A cross-sectional analysis of computerized general practice data was carried out in 43 practices in Scotland contributing to the Continuous Morbidity Recording project. The main outcome measures were odds ratios of being under GP review; receiving different classes of antihypertensive treatments [thiazides, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers]; and receiving other cardiovascular preventative treatments (statins and/or antiplatelets). RESULTS: Compared with males, female hypertensive patients were more likely to receive a thiazide and less likely to be given an ACE inhibitor, calcium channel blocker or secondary preventative treatment. Elderly hypertensive patients were less likely than the youngest patients to be under GP active review, more likely to be on a thiazide, calcium channel blocker or antiplatelet treatment, and less likely to be on a statin. More deprived hypertensive patients were less likely to be under GP review, or to be on a thiazide or a statin, but were more likely to be on a calcium channel blocker or an antiplatelet drug than the most affluent group. CONCLUSIONS: Important gender, age and deprivation differences exist in three important components of the primary care treatment of hypertension in Scotland.

Coincidence of immune-mediated diseases driven by Th1 and Th2 subsets suggests a common aetiology. A population-based study using computerized general practice data.

BACKGROUND: The recent rise in the prevalence of immune-mediated diseases has been attributed to environmental factors such as a lack of microbial challenge, or dietary change, that deviate the overall balance between mutually antagonistic subsets of T helper (Th) cells. OBJECTIVE: An alternative proposal is that recent environmental changes have resulted in an immune system that is more likely to produce both Th1 and Th2 responses against benign antigens. The prediction of this hypothesis, that Th1 and Th2-mediated diseases are not mutually exclusive, and may be positively associated, is tested here in a whole population. METHODS: Data from General Practices participating in the Scottish Continuous Morbidity Recording (CMR) project were used to determine the coincidence of the major Th2-mediated atopic diseases; asthma, eczema and allergic rhinitis, with the Th1-mediated autoimmune conditions; type I diabetes, rheumatoid arthritis and psoriasis. We also identified the prescription rates of inhaled therapy for asthma in patients with Th1-mediated disease. RESULTS: There was a significant increase in the risk of presenting with a Th1-mediated autoimmune condition in patients with a history of allergic disease (standardized prevalence ratio (95% confidence interval) 1.28 (1.18-1.37)). Likewise, the standardized prevalence ratios of presenting with either eczema (1.67 (1.48-1.87)) or allergic rhinitis (1.22 (1.02-1.44)) were significantly increased in subjects with a history of Th1-mediated disease. There was a particularly strong association between current psoriasis and current eczema (standardized prevalence ratio ofpsoriasis in subjects with eczema 2.88, 95% confidence interval (CI) 2.38-3.45). There was also a significant increase in prescriptions for inhaled asthma therapy in patients with Th1 disease. CONCLUSION: It is concluded that Th1- and Th2-mediated diseases are significantly associated in a large General Practice population. This finding supports the proposal that autoimmune and atopic diseases share risk factors that increase the propensity of the immune system to generate both Th1- and Th2-mediated inappropriate responses to non-pathological antigens.

Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.

Enhancement of innate immunity against Mycobacterium avium infection by immunostimulatory DNA is mediated by indoleamine 2,3-dioxygenase.

Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course of Mycobacterium avium infection. M. avium growth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-alpha/beta), and IFN-gamma. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.

Induction of indoleamine 2,3-dioxygenase in vivo by IFN-con1.

Indoleamine 2,3-dioxygenase (IDO) activity as determined by increases in serum kynurenine was measured in a group of hepatitis C patients treated with consensus interferon (IFN-con1). Kynurenine levels increased significantly within 2 days of initiation of treatment but returned to normal values by week 4 after treatment. Although IDO is normally induced by IFN-gamma, no such IFN was detected by ELISA or biologic assays. Thus, consensus IFN induces low levels of IDO in vivo without an IFN-gamma intermediate.

The role of the health care leader. Past, present, future.

Strong, effective communication may be the single most important key to success for any type of executive. Leaders of health care organizations must be able to promote ideas that others will implement, help staff see the value of their work, and have the vision to overcome limitations that stand in the way of progress.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon.

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

An indoleamine 2,3-dioxygenase-negative mutant is defective in stat1 DNA binding: differential response to IFN-gamma and IFN-alpha.

We have previously reported the isolation of mutant cell lines from the human carcinoma line ME180 that are resistant to the antiproliferative effect of interferon-gamma (IFN-gamma). These cell lines were defective in the induction of indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan catabolism. One of these cell lines, 3B6A, was chosen for further study. This cell line was also defective in the ability of IFN-gamma to protect against vesicular stomatitis virus (VSV) infection. However it maintained a normal antiviral response to IFN-alpha. A promoter-chloramphenicol acetyltransferase (CAT) construct containing the promoter region of IDO, which includes IFN-gamma activation site (GAS), IFN-stimulated response element-1 (ISRE-1), and ISRE-2 regions, was not expressed in 3B6A in the presence of IFN-gamma, indicating that the defect was likely to be in either Stat1 or IFN regulatory factor-1 (IRF-1), transcription factors known to bind to these cis-acting sequences. The induction of other IFN-gamma-inducible genes, such as tryptophanyl-tRNA synthetase (hWRS), was also affected. Electrophoretic mobility shift assays (EMSA) comparing nuclear extracts from parental and mutant cells indicated that Stat1 from the mutant did not bind to GAS sequences. However, Western blot analysis indicated that Stat1 protein was present. This IDO-negative phenotype can be reversed by transfection with a Stat1 expression vector. DNA sequencing of the Stat1 cDNA from wild-type and 3B6A cells indicated that an amino acid change occurred in the Stat1 protein of the mutant at W573, a tryptophan conserved in all known Stat proteins. We hypothesize that a change in this region of the Stat protein affects the response to IFN-gamma but not to IFN-alpha.

Analysis of transcription factors regulating induction of indoleamine 2,3-dioxygenase by IFN-gamma.

IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma. This induction was reduced by cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha by IFN-gamma were reduced in 3B6A cells, and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Statla. Electrophoretic mobility shift assays indicated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.

Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation.

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2, 3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-gamma. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-gamma. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl -tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.

The effects of interferon on the expression of human papillomavirus oncogenes.

The effects of interferon (IFN)-alpha, IFN-beta and IFN-gamma on human papillomavirus (HPV) oncogene expression were studied in various cervical carcinoma cell lines containing integrated copies of either HPV type 16 or HPV type 18. The levels of E6 and E7 transcripts were examined 6 h and 30 h after treatment with IFN. In HeLa cells, all three classes of IFNs effected a decrease in the level of HPV-18 E6 and E7 transcripts. On the other hand, none of the IFNs altered the level of these transcripts in C-4II cells. Only IFN-gamma decreased the level of HPV-16 E6 and E7 transcripts in CaSki and HPK1A cells, while IFN-gamma actually increased the level of these transcripts in SiHa cells. This differential IFN regulation of HPV expression in various cervical cancer cell lines may account for the contradictory clinical results observed after treatment of cervical cancer with IFN.

Respiratory morbidity in primary care. A population based study, using practices from the Scottish Continuous Morbidity Recording Research Database.

OBJECTIVE: To elucidate the patterns and period prevalences of respiratory disease with special reference to asthma (including wheezing) in view of its increasing reported prevalence. DESIGN: Observational study based on prospectively entered data. SETTING/SUBJECTS: Fifty five Continuous Morbidity Recording (CMR) practices with 290,000 patients located throughout Scotland. RESULTS: Respiratory problems accounted for a large proportion (17%) of total general practice workload. Upper respiratory tract infections were the commonest presentation in pre-school children, followed by asthma but with an ever increasing proportion of consultations for bronchitis and lower respiratory tract infections with advancing adult age. There was no significant correlation between deprivation and the incidence of asthma. CONCLUSIONS: Observed rates and patterns of disease for CMR practices, were similar to previously reported studies. The large number of presentations by patients in early childhood with minor respiratory illnesses and in particular upper respiratory tract infections are likely to reflect a heightened level of parental anxiety where interpretation of clinical signs and separation of simple and significant illness can be difficult. CMR has also been shown to be of use in helping to investigate links between deprivation and disease incidence or severity. Potential uses for CMR include the study of whole population morbidity and utilisation of primary care services.

Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase.

A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast.