Control of arbovirus infections by a coordinated response: West Nile Virus in England and Wales

Although there is no recognized transmission of human arboviral infections in the UK, concerns about the possible spread of West Nile virus (WNV) have precipitated coordinated activities around both surveillance and response. The Department of Health has chaired a UK WNV task force since the end of 2000. This is a multidisciplinary group of senior representatives from Agencies and Government Departments involved in human and animal health, entomology and academic departments. Activities include surveillance for WNV infections in humans, and in dead birds, mosquitoes and horses. All have been negative for WNV. A WNV contingency plan was produced in 2004, and this could be used as a generic plan for an effective and coordinated response in the event of the emergence of a new vector-borne zoonotic infection.     

The influence of method-related partner violence on covert pill use and pill discontinuation among women living in La Paz, El Alto and Santa Cruz, Bolivia

Intimate partner violence is widespread worldwide. While assumed to impact women's ability to use contraceptive methods, few data are available to support this claim. In this study, eight focus group discussions were conducted to guide questionnaire development and to provide contextual information. Participants were women who were currently using the pill and women who had used the pill previously. In addition, 300 women were interviewed who initiated oral contraceptive pill use between December 1995 and April 1996. Participants were interviewed 3-6 months later to investigate the role intimate partner violence played in covert pill use and pill discontinuation. Special study procedures for asking women questions about violence were employed. Nineteen per cent of the women interviewed were using the pill covertly. The odds of covert pill use were four times higher in El Alto and La Paz than in Santa Cruz. Women who used the pill covertly were more likely to have experienced method-related partner violence (OR = 21.27) than women whose partners knew of their pill use. One-third of the women had discontinued pill use at the time of the interview. In the final multivariate analysis, having experienced side-effects (OR = 2.37) was a significant predictor of pill discontinuation and method-related partner violence was marginally predictive (OR = 1.91; 95% CI 1.0-3.66). While efforts are ongoing to incorporate men into family planning programmes, some male partners oppose, and in some situations violently oppose, contraceptive use. The needs of women with these types of partners must not be overlooked.     

Pharmacologic inhibitors of extracellular signal-regulated kinase (ERKs) and c-Jun NH(2)-terminal kinase (JNK) decrease glutathione content and sensitize human promonocytic leukemia cells to arsenic trioxide-induced apoptosis

Treatment with 1-4 microM As(2)O(3) slightly induced apoptosis in U-937 human promonocitic leukemia cells. This effect was potentiated by co-treatment with MEK/ERK (PD98059, U0126) and JNK (SP600125, AS601245) inhibitors, but not with p38 (SB203580, SB220025) inhibitors. However, no potentiation was obtained using lonidamine, doxorubicin, or cisplatin instead of As(2)O(3). Apoptosis potentiation by mitogen-activated protein kinase (MAPK) inhibitors involved both the intrinsic and extrinsic executionary pathways, as demonstrated by Bax activation and cytochrome c release from mitochondria, and by caspase-8 activation and Bid cleavage, respectively; and the activation of both pathways was prevented by Bcl-2 over-expression. Treatment with MEK/ERK and JNK inhibitors, but not with p38 inhibitors, caused intracellular glutathione (GSH) depletion, which was differentially regulated. Thus, while it was prevented by N-acetyl-L-cysteine (NAC) in the case of U0126, it behaved as a NAC-insensitive process, regulated at the level of DL-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, in the case of SP600125. The MEK/ERK inhibitor also potentiated apoptosis and decreased GSH content in As(2)O(3)-treated NB4 human acute promyelocytic leukemia (APL) cells, but none of these effects were produced by the JNK inhibitor. MEK/ERK and JNK inhibitors did not apparently affect As(2)O(3) transport activity, as measured by intracellular arsenic accumulation. SP600126 greatly induced reactive oxygen species (ROS) accumulation, while BSO and U0126 had little or null effects. These results, which indicate that glutathione is a target of MAP kinases in myeloid leukemia cells, might be exploited to improve the antitumor properties of As(2)O(3), and provide a rationale for the use of kinase inhibitors as therapeutic agents.     

Apoptotic cells, through transforming growth factor-beta, coordinately induce anti-inflammatory and suppress pro-inflammatory eicosanoid and NO synthesis in murine macrophages

Apoptotic cells are rapidly engulfed by adjacent tissue cells or macrophages before they can release pro-inflammatory/proimmunogenic intracellular contents. In addition, recognition of the apoptotic cells is actively anti-inflammatory and anti-immunogenic with generation of anti-inflammatory mediators such as transforming growth factor-beta (TGF-beta) and anti-inflammatory eicosanoids. Here, we have investigated the role played by the induction of TGF-beta in the coordinate expression of anti-inflammatory eicosanoids or peroxisome proliferator-activated receptor-gamma and in the suppression of pro-inflammatory lipid mediators and nitric oxide (NO). By use of a dominant negative TGFbetaII receptor, TGF-beta signaling was blocked, and its participation in the consequences of apoptotic cell stimulation was determined. The induction of TGF-beta itself could be attributed to exposed phosphatidylserine on the apoptotic cells, which therefore appears to drive the balanced inflammatory mediator responses. Arachidonic acid release, COX-2, and prostaglandin synthase expression were shown to be significantly dependent on the TGF-beta production. On the other hand, a requirement for TGF-beta was also shown in the inhibition of thromboxane synthase and thromboxanes, of 5-lipoxygenase and sulfidopeptide leukotrienes, as well as of inducible nitric-oxide synthase and NO. TGF-beta-dependent induction of arginase was also found and would further limit the NO generation. Finally, apoptotic cells stimulated production of 15-lipoxygenase and 15-hydroxyeicosatetraenoic acid, a potentially anti-inflammatory pathway acting through peroxisome proliferator-activated receptor-gamma, and lipoxin A(4) production, which were also up-regulated by a TGF-beta-dependent pathway in this system. These results strongly suggest that the apoptotic cell inhibition of pro-inflammatory mediator production is pleiotropic and significantly dependent on the stimulation of TGF-beta production.     

Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol

Previous work has shown that IL-16/CD4 induces desensitization of both CCR5- and CXCR4-induced migration, with no apparent effect on CCR2b or CCR3. To investigate the functional relationship between CD4 and other chemokine receptors, we determined the effects of IL-16 interaction with CD4 on CXCR3-induced migration. In this study we demonstrate that IL-16/CD4 induced receptor desensitization of CXCR3 on primary human T cells. IL-16/CD4 stimulation does not result in surface modulation of CXCR3 or changes in CXCL10 binding affinity. This effect does require p56(lck) enzymatic activity and the presence of CCR5, because desensitization is not transmitted in the absence of CCR5. Treatment of human T cells with methyl-beta-cyclodextrin, a cholesterol chelator, prevented the desensitization of CXCR3 via IL-16/CD4, which was restored after reloading of cholesterol, indicating a requirement for intact cholesterol. These studies demonstrate an intimate functional relationship among CD4, CCR5, and CXCR3, in which CCR5 can act as an adaptor molecule for CD4 signaling. This process of regulating Th1 cell chemoattraction may represent a mechanism for orchestrating cell recruitment in Th1-mediated diseases.     

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.     

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.     

Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic alpha-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.     

Preservation of micro-organisms by drying; a review

The preservation of micro-organisms by different drying methodologies has been used for decades. Freeze drying in particular is the preferred method for transporting and storing vast culture collections of micro-organism strain types. The literature on drying and preserving micro-organisms is extensive, but is often specific to one particular strain. This review attempts to draw some similar concepts and findings together in one paper, to compare different drying techniques, with specific reference to microorganisms. The main topics covered are cell growth phases and concentration, inducing drying tolerance in microbial cells, drying methods, rehydration of dried cells and packaging and storage conditions. Also, particular attention has been paid to the use of freeze drying and the protective matrices used to improve microbial cell viability after drying.