Influence of ursodeoxycholic acid on blood glucose, insulin and GLP-1 in rats with liver fibrosis induced by bile duct ligation.

BACKGROUND: The prevalence of impaired glucose tolerance and diabetes is much higher in people with cirrhosis than that in the general population. However, there are inadequate concrete guidelines for the management of diabetes in these patients, particularly in the early stage. Bile aids (BAs) have been found to exert hormone-like functions in the control of lipid and glucose metabolism. We studied the effect of ursodeoxycholic acid (UDCA) on glucose levels in rats with cirrhosis induced by bile duct ligation (BDL). METHODS: SD rats were divided into three groups: sham operation (Group A); BDL (Group B), and UDCA plus BDL (Group C). After 4 weeks, oral glucose tolerance tests were performed. Serum biochemical parameters and the levels of glucose, insulin, and glucagon-like peptide 1 (GLP-1) were measured. Histopathology of the liver and islet was observed. The gene expression of cholesterol 7α-hydroylase (CYP7A1), microsomal oxysterol 7a-hydroxylase (CYP7B1) in the liver, and Takeda G-protein-coupled receptor-5 (TGR5) in the intestine was determined by real-time PCR. RESULTS: Compared with Group A, fasting glucose and 1-h and 2-h postprandial glucose levels increased slightly (all P > 0.05), 2-h postprandial insulin levels increased significantly (P < 0.05), 15 min postprandial GLP-1 levels decreased (P < 0.05) in Group B. Compared with Group B, fasting glucose and 1-h postprandial glucose levels decreased (all P < 0.05), 2-h postprandial insulin levels decreased (P < 0.01), and 15 min postprandial GLP-1 levels increased (P < 0.05) in Group C. After UDCA intervention, liver fibrosis induced by BDL was alleviated, and the islet areas were increased (P < 0.05). Compared with Group A, the mRNA expression of CYP7A1 and CYP7B1 in the liver increased, and the mRNA expression of TGR5 in the intestine decreased in Group B (all P < 0.05). Compared with Group B, the mRNA expression of CYP7A1 and CYP7B1 in the liver decreased, and TGR5 in the intestine increased in Group C (P < 0.05). CONCLUSIONS: After 4 weeks of BDL, the rats developed liver fibrosis and abnormal glucose metabolism. UDCA administration improved liver fibrosis, increased islet area, decreased glucose levels, inhibited genes in BA synthesis, enhanced TGR5 gene expression in the intestine, and further improved islet function.

The Significance of EEG Alpha Oscillation Spectral Power and Beta Oscillation Phase Synchronization for Diagnosing Probable Alzheimer Disease.

Alzheimer disease (AD) is the most common cause of dementia in geriatric population. At present, no effective treatments exist to reverse the progress of AD, however, early diagnosis and intervention might delay its progression. The search for biomarkers with good safety, repeatable detection, reliable sensitivity and community application is necessary for AD screening and early diagnosis and timely intervention. Electroencephalogram (EEG) examination is a non-invasive, quantitative, reproducible, and cost-effective technique which is suitable for screening large population for possible AD. The power spectrum, complexity and synchronization characteristics of EEG waveforms in AD patients have distinct deviation from normal elderly, indicating these EEG features can be a promising candidate biomarker of AD. However, current reported deviation results are inconsistent, possibly due to multiple factors such as diagnostic criteria, sample sizes and the use of different computational measures. In this study, we collected two neurological tests scores (MMSE and MoCA) and the resting-state EEG of 30 normal control elderly subjects (NC group) and 30 probable AD patients confirmed by Pittsburgh compound B positron emission tomography (PiB-PET) inspection (AD group). We calculated the power spectrum, spectral entropy and phase synchronization index features of these two groups' EEG at left/right frontal, temporal, central and occipital brain regions in 4 frequency bands: δ oscillation (1-4 Hz), θ oscillation (4-8 Hz), α oscillation (8-13 Hz), and ß oscillation (13-30 Hz). In most brain areas, we found that the AD group had significant differences compared to NC group: (1) decreased α oscillation power and increased θ oscillation power; (2) decreased spectral entropy in α oscillation and elevated spectral entropy in ß oscillation; and (3) decrease phase synchronization index in δ, θ, and ß oscillation. We also found that α oscillation spectral power and ß oscillation phase synchronization index correlated well with the MMSE/MoCA test scores in AD groups. Our study suggests that these two EEG features might be useful metrics for population screening of probable AD patients.

The Validation of Multifactor Model of Plasma Aß 42 and Total-Tau in Combination With MoCA for Diagnosing Probable Alzheimer Disease.

Alzheimer disease (AD) has an insidious onset and heterogeneous clinical symptoms. The well-accepted biomarkers for clinical diagnosis of AD include ß-amyloid (Aß) deposition and pathologic tau level within cerebral spinal fluid (CSF) and imaging AD pathology such as positive emission tomography (PET) imaging of the amyloid-binding agent Pittsburgh compound B (PET-PiB). However, the high expense and invasive nature of these methods highly limit their wide usage in clinic practice. Therefore, it is imperious to develop less expensive and invasive methods, and plasma biomarkers are the premium targets. In the current study, we utilized a single-blind comparison method; all the probable AD cases met the core clinical National Institute on Aging and Alzheimer's Association (NIA-AA) criteria and validated by PET-PiB. We used ultrasensitive immunomagnetic reduction (IMR) assays to measure plasma Aß 42 and total-tau (t-tau) levels, in combination with different variables including Aß42 × t-tau value, Montreal Cognitive Assessment (MoCA), and Mini Mental State Examination (MMSE). We used logistic regression to analyze the effect of all these variables in the algorism. Our results showed that (1) plasma Aß42 and t-tau are efficient biomarkers for AD diagnosis using IMR platform, whereas Aß42 × t-tau value is more efficient for discriminating control and AD; (2) in the control group, Aß42 level and age demonstrated strong negative correlation; Aß42 × t-tau value and age demonstrated significant negative correlation; (3) in the AD group, t-tau level and MMSE score demonstrated strong negative correlation; (4) using the model that Aß42, Aß42 × t-tau, and MoCA as the variable to generate receiver operating characteristic (ROC) curve, cutoff value = 0.48, sensitivity = 0.973, specificity = 0.982, area under the curve (AUC) = 0.986, offered better categorical efficacy, sensitivity, specificity, and AUC. The multifactor model of plasma Aß42 and t-tau in combination with MoCA can be a viable model separate health and AD subjects in clinical practice.

Catenovulum sediminis sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, strictly aerobic, rod-shaped and agar-hydrolysing bacterium, designated D2T, was isolated from a marine sediment sample collected from the coast of Weihai, China (37° 31' 59″ N 122° 03' 47″ E). The cells were motile by a lateral flagellum. Growth was observed at 10-42 °C, at pH 6.0-9.0 and with 0.5-8 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain D2T belonged to the genus Catenovulum, appearing closely related to Catenovulum agarivorans YM01T (96.3 % 16S rRNA gene sequence similarity) and Catenovulum maritimumQ1T (93.9 %). The dominant fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C10 : 0 3-OH. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, two phosphoaminolipids, two unknown lipids and three phospholipids. Ubiquinone 8 (Q-8) was found to be the major respiratory quinone. The DNA G+C content was 40.4 mol%. On the basis of genotypic, phenotypic and phylogenetic evidence, strain D2T is presented as a representative of a novel species of the genus Catenovulum, for which the name Catenovulum sediminis sp. nov. is proposed. The type strain is D2T (=KCTC 42869T=MCCC 1H00129T). An emended description of the genus Catenovulum is also provided.

Association between clusterin gene polymorphism rs11136000 and late-onset Alzheimer's disease susceptibility: A review and meta-analysis of case-control studies.

The present study aimed to evaluate the association between rs11136000 in clusterin (CLU) and late-onset Alzheimer's disease (LOAD) by meta-analysis. Several databases including PubMed, EMbase, CBMdisc and CMCC were searched for relevant case-control studies based on defined selection criteria. Odds ratios (OR) and 95% confidence interval (CI) of the rs11136000 genotype and allele distribution were analyzed with RevMan and Stata software. The control population and heterogeneity between populations were examined in the selected studies using the Hardy-Weinberg equilibrium. Overall OR among the frequencies of the genotype and allele in both patients with AD and controls was estimated using fixed or random effect models. The summary of the OR and 95% CI were then analyzed to obtain the effects across the studies. Publication bias was examined using a funnel plot, Egger's test and Begg's test, and a Fail-safe Number (Nfs). A total of 20 reports were used. The summary OR for studies in the Caucasian population with a frequency of TT+TC/CC genotype and T/C allele at rs11136000 locus in CLU were 0.79 (95% CI, 0.73-0.86; P<0.00001) and 0.87 (95% CI, 0.85-0.90; P<0.00001). The summary OR for the studies conducted in the Asian population were 0.90 (95% CI, 0.81-0.99; P=0.04) and 0.87 (95% CI, 0.81-0.93; P<0.0001). The summary OR in other mixed ethnic groups with regards to the frequency of T/C allele was 0.82 (95% CI, 0.68-0.99; P=0.04). These results demonstrated the presence of a statistically significant difference in LOAD susceptibility between individuals with the T allele CLU rs11136000 polymorphism and those without. The studies conducted in populations of African descent or Hispanics showed no statistically significant difference. Negligible publication bias was present, with Nfs being 750.604. In summary, polymorphism rs11136000 in the CLU gene may contribute to susceptibility to LOAD, and the presence of the T allele may reduce the risk of LOAD in Caucasian and Asian populations. However, no definitive association was found between the presence of the CLU rs11136000 polymorphism and LOAD in populations of African or Hispanic descent.

Physical activity as a protective factor against depressive symptoms in older Chinese veterans in the community: result from a national cross-sectional study.

BACKGROUND: Physical activity is generally considered to be effective in reducing the prevalence of depression and promoting remission of its symptoms. However, large-scale epidemiological research on this issue is lacking in older Chinese adults. We performed a nationwide epidemiological survey to determine the relationship between physical activity and depressive symptoms in older Chinese veterans in the community, with adjustment for potential confounders. METHODS: A cross-sectional study was conducted in a representative sample of 9,676 community-dwelling older Chinese veterans. Depressive symptoms were identified using the Center for Epidemiological Studies Depression Scale. Physical activity was self-reported using a one-year physical activity questionnaire. Information about covariates was obtained by questionnaire-based interview. Relationships between study variables and symptoms of depression were estimated using unadjusted and adjusted analyses. RESULTS: The median age was 82.29 (interquartile range 80.25-84.60) years. In total, 81.84% of the study participants engaged in physical activity that was predominantly light in intensity. In unadjusted analyses, physical activity was associated with a significantly decreased likelihood of depressive symptoms (5.43% versus 18.83%, P<0.0001). Multivariate logistic regression with adjustment and controlling for confounders, physical activity was still inversely associated with depressive symptoms and was the only independent protective factor (odds ratio 0.57, 95% confidence interval 0.44-0.72, P<0.0001) among the associated factors in this study. In a univariate general linear model, there was a significant difference in Center for Epidemiological Studies Depression Scale score between subjects participating in active physical activity and those who did not (F=59.07, P<0.0001). CONCLUSION: This study found an inverse relationship between physical activity and symptoms of depression in older Chinese veterans in the community. It was also indicated that the antidepressant effect of physical activity probably extended to the oldest-old, and the light-intensity physical activity was probably available for the same protective effect. This information could be used to devise further interventions to prevent or ameliorate symptoms of depression.

Cattle encephalon glycoside and ignotin injection improves cognitive impairment in APPswe/PS1dE9 mice used as multitarget anti-Alzheimer's drug candidates.

BACKGROUND: Cattle encephalon glycoside and ignotin injection (CEGI), a multitargeted neurotrophic drug, has been widely used in the treatment of central and peripheral nerve injuries, such as stroke, hypoxic ischemic encephalopathy, and diabetic neuropathy in the People's Republic of China. However, data regarding the effect of CEGI on Alzheimer's disease (AD) remain scarce. The present study aimed to investigate the effect of CEGI on learning and memory in an APPswe/PS1dE9 double-transgenic mouse model, a suitable animal model of AD, and elucidate its possible mechanisms. MATERIALS AND METHODS: Five-month-old APP/PS1 mice were intraperitoneally administered 6.6 mL/kg or 13.2 mL/kg of CEGI for 1 month. After 1 month of administration, all mice received Morris water maze training and a probe test. Mouse brain sections were detected by standard biochemical and immunohistochemical measures. RESULTS: CEGI treatment significantly improved the spatial learning and memory deficits and decreased cerebral amyloid-ß42 levels in brain homogenates of APP/PS1 mice. CEGI treatment elevated the activities of superoxide dismutase, and reduced the levels of malondialdehyde. CEGI attenuated neuronal damage in the hippocampus of APP/PS1 mice and upregulated protein and gene expression of Bcl-2 and the ratio of Bcl-2/Bax. CEGI treatment decreased the number of Iba1(+) activated microglia in the cortex of the APP/PS1 mice. CONCLUSION: Our results showed that CEGI prevents memory impairment, possibly by decreasing the amyloid-ß42 levels in APP/PS1 mice and inhibiting oxidative stress, apoptosis, and inflammation, making CEGI a promising therapeutic agent for AD.

Factors Associated with Performance-based Physical Function of Older Veterans of the PLAAF: A Pilot Study.

[Purpose] This study investigated the factors associated with performance-based physical function of older veterans of the People's Liberation Army Air Force of China (PLAAF). [Subjects and Methods] A cross-sectional survey of 146 older veterans of the PLAAF was carried out. Their physical function was measured using the Chinese Mini-Physical Performance Testing (CM-PPT). The demographics and health status (including physical measures, blood chemical tests, chronic diseases, and number of morbidities) were collected from health examination reports and computer records of case history. Cognition was measured using the Mini-Mental Status Examination (MMSE). [Results] In multiple linear regressions, age, MMSE, Parkinsonism, and chronic obstructive pulmonary disease were independently associated with CM-PPT, while previous stroke and albumin level reached borderline statistical significance. The association between the number of morbidities and CM-PPT was significant after adjustment for MMSE and demographics. The CM-PPT of low (0 or 1), medium (2 to 4) and high count (5 or more) morbidities were 11.3±3.9, 10.2±4.1, 6.1±3.8 respectively, and the difference among these three groups was significant. [Conclusion] Some modified conditions and the number of chronic diseases might be associated with the physical function of older veterans of the PLAAF.

[Comparative analysis of conventional pulmonary function test results in children with asthma or cough variant asthma].

OBJECTIVE: To compare the conventional pulmonary function test results of children with asthma or cough variant asthma (CVA). METHODS: A total of 140 children, who were diagnosed with asthma or CVA from May 2010 to May 2011, were divided into acute asthma attack (n=50), asthma remission (n=50) and CVA groups (n=40); 30 healthy children were included as a control group. The forced vital capacity (FVC), forced expiratory volume in one second (FEV1), peak expiratory flow (PEF), forced expiratory flow after 25% of vital capacity has been expelled (FEF25), forced expiratory flow after 50% of vital capacity has been expelled (FEF50), forced expiratory flow after 75% of vital capacity has been expelled (FEF75) and maximal midexpiratory flow (MMEF75/25) were measured. RESULTS: The mean percent predicted values of all the above indices were lower than 80% in the acute asthma attack group, with FEF50, FEF75 and MMEF75/25 declining markedly; the mean percent predicted values of FEF75 and MMEF75/25 were lower than 80% in the CVA group. All the pulmonary function indices in the acute asthma attack group were lower than those in the control group. The mean percent predicted values of FVC, FEV1, FEF25 and MMEF75/25 in the asthma remission and CVA groups were lower than in the control group. All the pulmonary function indices in the acute asthma attack group were lower than in the asthma remission and CVA groups, but there were no significant differences between the asthma remission and CVA groups. CONCLUSIONS: There is small and large airway dysfunction, particularly small airway dysfunction, in children with acute asthma attack. Children with CVA present mainly with mild small airway dysfunction, as do those with asthma in remission.

Dysregulation of p38 and MKP-1 in response to NOD1/TLR4 stimulation in sarcoid bronchoalveolar cells.

RATIONALE: Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. OBJECTIVES: To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. METHODS: Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. MEASUREMENTS AND MAIN RESULTS: BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. CONCLUSIONS: Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

STAT3 tyrosine phosphorylation is critical for interleukin 1 beta and interleukin-6 production in response to lipopolysaccharide and live bacteria.

Both interleukin 1 beta (IL-1beta) and interleukin-6 (IL-6) are pro-inflammatory cytokines that play a major role in inflammatory diseases as well as cancer. In this work we investigated the signaling pathway involving lipopolysaccharide (LPS)-mediated IL-1beta and IL-6 production in murine macrophage cell lines and primary macrophages. We show that in response to LPS, the JAK/STAT pathway is activated, leading to tyrosine phosphorylation at residue 705 on STAT3 and at residue 701 on STAT1, respectively. A newly developed STAT3 specific inhibitor (stattic) blocked LPS-mediated STAT3 tyrosine phosphorylation and led to inhibition of LPS-mediated IL-1beta and IL-6 production but not TNF-alpha production. Knockdown of STAT3 expression via small interfering RNA (siRNA) decreased the level of STAT3 expression in Raw 264.7 cells and decreased STAT3 tyrosine phosphorylation in response to LPS treatment. Quantitative real time PCR and Western analysis of cells treated with inhibitor or STAT3 siRNA after LPS treatment showed a significant reduction of IL-1beta and IL-6 mRNA and protein compared to cells treated with LPS alone. Moreover stattic abrogated IL-1beta formation in response to extracellular bacteria Staphylococcus aureus and Escherichia coli in murine peritoneal macrophages. This inhibition did not affect caspase-1 activation. These results highlight the complex role of STAT3 in cytokine production and the key role of STAT3 tyrosine phosphorylation in IL-1beta and IL-6 production in response to inflammation.

ST2 is essential for Th2 responsiveness and resistance to pseudomonas aeruginosa keratitis.

PURPOSE: To elucidate the role of ST2, a member of the TLR/IL-1R (TIR) superfamily, in protecting against Pseudomonas aeruginosa keratitis in BALB/c mice. METHODS: ST2 mRNA and protein expression levels were tested by real-time PCR and Western-blot in C57BL/6 (B6; susceptible) versus BALB/c (resistant) mice before and after P. aeruginosa (strain 19660; American Type Culture Collection, Philadelphia, PA) challenge. Infected BALB/c mice also were tested after subconjunctival injection with recombinant murine (rm)ST2 or PBS. Disease was monitored by clinical score, slit lamp, bacterial plate count, a myeloperoxidase (MPO) assay to measure polymorphonuclear neutrophil (PMN) infiltrate, real-time RT-PCR, and ELISA. RESULTS: ST2 mRNA and protein were constitutively expressed in the uninfected normal corneas of both mouse groups. ST2 levels in the cornea of BALB/c compared with B6 mice were elevated significantly at 1 to 3 days post infection (PI), peaked at 3 and decreased at 5 days PI. BALB/c mice treated with rmST2 showed increased corneal opacity and perforation (at 5 days PI) when compared with PBS controls. rmST2- versus PBS-injected mice exhibited increased bacterial load, PMN infiltrate, and higher corneal mRNA levels for IL-1beta, MIP-2, IL-6, IL-1R1, and Th1-type cytokine such as IFN-gamma. Protein levels for IL-1beta, MIP-2, and IL-6 also were significantly upregulated, whereas the Th2 cytokines IL-4 (mRNA), IL-5 (mRNA), and IL-10 (mRNA and protein) were significantly reduced. CONCLUSIONS: ST2 is critical in resistance to P. aeruginosa keratitis, functioning to reduce corneal infection (bacterial load) and inflammation by negatively regulating proinflammatory cytokines and inhibiting type-1 immunity, but upregulating type-2 cytokine production, particularly IL-10.

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice.

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.

TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis.

PURPOSE: To determine the role of Toll-like receptor 4 (TLR4) in Pseudomonas aeruginosa (P. aeruginosa) keratitis in resistant (cornea-healing) BALB/c mice. METHODS: Corneal TLR4 mRNA levels were tested by real-time PCR in BALB/c mice before and after infection. Clinical score, slit lamp, histopathology, bacterial counts, and polymorphonuclear neutrophil (PMN) quantitation were performed in the infected cornea of TLR4-deficient (TLR4(lps-d)) and wild-type BALB/c mice. mRNA for IL-1beta, MIP-2, IFN-gamma, IL-18, inducible nitric oxide synthase (iNOS), and beta-defensin-2 levels were measured by real-time PCR. Protein levels for IL-1beta, MIP-2, and IFN-gamma were tested by ELISA. RESULTS: In resistant BALB/c mice, TLR4 mRNA expression was significantly upregulated in the cornea after P. aeruginosa infection. In contrast, TLR4-deficient mice were susceptible to infection with P. aeruginosa and showed increased corneal opacity, PMN infiltration, bacterial counts, and perforated infected corneas. After infection, TLR4-deficient mice also showed increased mRNA expression of proinflammatory cytokines (IL-1beta and MIP-2) and type-1-associated cytokines (IFN-gamma and IL-18) when compared with wild-type BALB/c mice. ELISA analyses showed that IL-1beta, MIP-2, and IFN-gamma protein levels also were significantly upregulated in the cornea of TLR4-deficient versus wild-type mice. In contrast, levels of iNOs and beta-defensin-2 were significantly decreased in TLR4-deficient compared with wild-type mice. CONCLUSIONS: TLR4 is critical in host resistance to P. aeruginosa, as its deficiency results in increased PMN infiltration and proinflammatory cytokine production, decreased iNOs and beta-defensin-2 production, impaired bacterial killing, and a susceptible phenotype.

SIGIRR promotes resistance against Pseudomonas aeruginosa keratitis by down-regulating type-1 immunity and IL-1R1 and TLR4 signaling.

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible Th1 responder C57BL/6 (B6), but not resistant Th2 responder (BALB/c) mice. To determine whether single Ig IL-1R-related molecule (SIGIRR) played a role in resistance, mRNA and protein expression levels were tested. Both were constitutively expressed in the cornea of the two mouse groups. A disparate mRNA and protein expression pattern was detected in the cornea of BALB/c vs B6 mice after infection. SIGIRR protein decreased significantly in BALB/c over B6 mice at 1 day postinfection. Thus, BALB/c mice were injected with an anti-SIGIRR Ab or IgG control. Anti-SIGIRR Ab over control-treated mice showed increased corneal opacity, stromal damage, and bacterial load. Corneal mRNA levels for IL-1beta, MIP-2, IL-1R1, TLR4, IL-18, and IFN-gamma and protein levels for IL-1beta and MIP-2 also were significantly up-regulated in anti-SIGIRR Ab over control mice, while no changes in polymorphonuclear cell number, IL-4, or IL-10 mRNA expression were detected. To further define the role of SIGIRR, RAW264.7 macrophage-like cells were transiently transfected with SIGIRR and stimulated with heat-killed P. aeruginosa or LPS. SIGIRR transfection significantly decreased mRNA levels for IL-1R1, TLR4, and type 1 immune response-associated cytokines (IL-12, IL-18, and IFN-gamma) as well as proinflammatory cytokines IL-1beta and MIP-2 protein expression. SIGIRR also negatively regulated IL-1 and LPS, but not poly(I:C)-mediated signaling and NF-kappaB activation. These data provide evidence that SIGIRR is critical in resistance to P. aeruginosa corneal infection by down-regulating type 1 immunity, and that it negatively regulates IL-1 and TLR4 signaling.

Shuttle vectors for Candida albicans: control of plasmid copy number and elevated expression of cloned genes.

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20-40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.

Fungicidal activity of fluconazole against Candida albicans in a synthetic vagina-simulative medium.

Fluconazole (FLZ) has emerged as a highly successful agent in the management of systemic infections of Candida. Cure rates for symptomatic candidiasis following single 150-mg FLZ dose therapy exceed 90%. In vitro, however, FLZ is fungistatic only in a narrow pH range and is not effective at vaginal pH, 4.2. This study evaluated the effect of FLZ on Candida albicans under in vitro conditions resembling the vaginal microenvironment, using vagina-simulative medium (VS). We found that FLZ was fungicidal for C. albicans in VS, but not in other media at the same pH, 4.2. In VS, FLZ was fungicidal at concentrations of >/=8 micro g/ml and reduced viability by greater than 99.9%. Analysis of the components of VS indicated that 17 mM acetic acid, a concentration achieved in the vagina, was responsible for the synergistic, fungicidal effect. This effect was not seen at neutral pH. Other substrates were not effective substitutes for acetic acid; however, short-chained carboxylic acids, glyoxylate and malonate, were effective. Most strains of C. albicans that were resistant to FLZ under standard conditions were killed by FLZ plus acetate. Other species of Candida were also killed, except C. krusei and C. glabrata. This study shows that FLZ has fungicidal activity for Candida species under in vitro conditions that mimic the vaginal microenvironment. This raises the possibility that FLZ may also have fungicidal effects during treatment of vaginal candidiasis. Elucidating the mechanism by which FLZ and acetate interact may disclose vulnerable pathways that could be exploited in drug development.