Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef.

A number of outbreaks of Escherichia coli O157:H7 infections involving beef have been reported. Options for controlling bacterial pathogens in raw foods are limited, but one is to use bacteriophages (phages). We describe the isolation and characterisation of phage FAHEc1, which infects E. coli O157, and its ability to kill its host in vitro and on beef. The phage belonged to the family Myoviridae and lysed 28 of 30 E. coli O157 (:H7, :HNM and :H not specified) isolates, only one other non-O157 E. coli serotype (O162:H7), and none of the other 13 bacterial species tested. The phage did not contain stx1, stx2, eae or ehxA virulence genes as assessed by PCR. An approximate 4 log10 inactivation of E. coli O157:H7 occurred at 5 °C in the presence of phage FAHEc1 at >107 PFU/ml in broth in vitro. On thinly sliced beef pieces incubated at 37 °C, a > 2.7 log10 reduction occurred with 3.2 × 107 PFU/4 cm² meat piece. At lower phage concentrations (10³-104 PFU/4 cm² piece) phage replication occurred on beef at 37 °C. When the phage was applied to beef pieces under conditions simulating hot boning and conventional carcass cooling, inactivation of E. coli O157:H7 of approximately 2 log10 was measured under optimal conditions with phages applied at 3.2 × 107 PFU/4 cm² meat piece.

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork.

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.

What is the Revised Fear Survey Schedule for Children measuring?

The Fear Survey Schedule for Children-Revised (FSSC-R) is a widely used self-report questionnaire that purports to measure the number of fears and the overall level of fearfulness in children. A number of studies have shown that the ten most common childhood fears can be found on the Danger and Death subscale of the FSSC-R, with upwards of 50% of children endorsing such fears. However, some researchers (e.g., H. McCathie & S.H. Spence, 1991; Behaviour Research and Therapy, 29, 495-502) have questioned the validity of these findings, suggesting that these items do not reflect actual childhood fears that children have or experience on a daily or regular basis. Rather, they suggest that children are responding to these fear items as if they were actually occurring to them in the here and now. The current study examined the occurrence of five Danger and Death fears from the FSSC-R (i.e., "Not being able to breathe", "Being hit by a car or truck", "Falling from high places", "Bombing attacks or being invaded", and "Fire or getting burned") in a sample of normal school children aged eight to 12 years (N=102). More specifically, we used three different methods to asses these fears: (1). prevalence as determined by the standard FSSC-R procedure, (2). prevalence as determined by a fear list procedure, and (3). actual occurrence or prevalence of these fears in the past week, as determined by a diary method. Results indicated that while these fears ranked high when using the standard FSSC-R procedure, they were considerably less common when using the fear list procedure, and had a low probability of actual occurrence on a daily basis, as well as possessing a short duration and low intensity. Implications for the assessment of fears and the use of self-report measures like the FSSC-R are briefly discussed.

Autologous lymphocyte-monocyte co-culture increases NMR-visible and cytoplasmic lipids in the absence of increased markers of lymphocyte activation.

Alterations in nuclear magnetic resonance (NMR)-visible lipid, morphometric lipid volume fraction, distribution of subcellular lipid droplets and activation antigen expression were examined in human peripheral blood lymphocytes, activated using phorbol myristate acetate (PMA) and ionomycin or by co-culture with autologous monocytes. PMA/Ionomycin treatment caused significant time-dependent increases in mobile lipid and in oil red O-positive lipid droplets that were accompanied by lymphocyte proliferation and increases in activation antigens, CD25, CD69 and CD71. Co-culture of lymphocytes and monocytes also induced significant increases in NMR-visible lipid signals and cytoplasmic lipid droplets, but in contrast, no correspondent increases in activation antigens were observed. Strong correlations were observed between the intensity of the NMR signal and the percentage of total cells containing lipid droplets (r=0.95) and the morphometric lymphocyte lipid volume fraction (r=0.80), indicating that the droplets were the source of the mobile lipid signal. Lipid droplets in PMA/Ionomycin-treated cells were evenly distributed throughout the population, but in co-cultures, only lymphocytes in close proximity to monocytes with lipid droplets contained oil red O-positive lipid. This data shows that the NMR-visible mobile lipid signal observed in lymphocytes co-cultured with monocytes is not directly dependent on either proliferation or the upregulation of activation antigens, similar to the previously observed response of T cells exposed to antibodies to the T cell receptor.

School refusal: epidemiology and management.

School refusal is differentiated from other attendance problems such as truancy and school withdrawal. It is characterised by the child's emotional upset at the prospect of going to school, parental awareness of and antipathy toward the problem, and an absence of significant antisocial behaviour in the child. The child's emotional upset is frequently associated with an anxiety disorder, but it may also be associated with a mood disorder. School refusal affects approximately 1% of school children across the primary and secondary school levels. Severe and prolonged school refusal jeopardises the young person's social, emotional and academic development, and may be associated with mental health problems in adulthood. A first step in management involves efficient identification and the assessment of contributing and maintaining factors. Clinical outcome studies support the efficacy of cognitive behavioural therapy (CBT). The psychosocial approach encompassed in CBT incorporates anxiety management training with the young person, behaviour management training with parents and consultation with school personnel. Pharmacological treatments are commonly employed although empirical support for their use is limited. Tricyclic antidepressants and selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitors are the more commonly used agents, with the latter having fewer associated adverse effects. It is suggested that the first line of treatment should be CBT, with simultaneous or subsequent pharmacological treatment contingent upon the response to CBT.

Transcriptional regulation of major histocompatibility complex class I by flavivirus West Nile is dependent on NF-kappaB activation.

Infection by the flavivirus West Nile (WNV) is associated with a virus-specific increase of major histocompatibility complex class I (MHC-I) molecules on the cell surface of diploid vertebrate cells. The increased MHC-I cell surface expression is functional and is associated with increased susceptibility to secondary WNV-immune and alloimmune cytotoxic T cells. WNV-induced up-regulation of cell surface MHC-I expression is associated with NF-kappaB activation and increased transcription of MHC-I mRNA. WNV infection increases luciferase activity of RAWa4 long terminal repeat (LTR) cells, which are transfected stably with a plasmid containing 2 NF-kappaB binding sites, the human immunodeficiency virus LTR linked to a luciferase reporter gene. The NF-kappaB-induced complexes are a p50/p65 heterodimer and another faster migrating species containing p50 homodimers. WNV-induced activation of NF-kappaB and the up-regulation of MHC-I were blocked by the protein kinase C inhibitor H-7 and salicylate, both of which block phosphorylation of inhibitor kappaB.

Interleukin-1beta but not tumor necrosis factor is involved in West Nile virus-induced Langerhans cell migration from the skin in C57BL/6 mice.

Langerhans cells are bone marrow-derived epidermal dendritic cells. They migrate out of the epidermis into the lymphatics and travel to the draining lymph nodes where they are responsible for the activation of T cells in the primary immune response. Tumor necrosis factor and interleukin-1beta, have previously been shown to be responsible for Langerhans cell migration in response to contact sensitizers in BALB/C mice; however, which cytokines are responsible for mediating Langerhans cell migration in response to a replicating cutaneously acquired virus such as the West Nile Virus, are not known. We have devised a method for identifying Langerhans cells in the draining lymph nodes using E-cadherin labeling and flow cytometry. We infected tumor necrosis factor-deficient gene knockout mice (tumor necrosis factor-/-) intradermally with West Nile Virus and found that levels of Langerhans cell emigration and accumulation in the draining lymph nodes were similar to wild-type C57BL/6 mice. This was borne out by the finding that high levels of systemic neutralizing anti-tumor necrosis factor antibody failed to inhibit the migration of Langerhans cells from the epidermis and their accumulation in the draining lymph nodes in wild-type C57BL/6 mice. In West Nile Virus-infected, tumor necrosis factor-/- mice treated with systemic neutralizing anti-interleukin-1beta antibodies, however, migration of Langerhans cells from the epidermis and their accumulation in the draining lymph nodes were significantly inhibited compared with control antibody-treated, infected animals. The results indicate that Langerhans cell migration, accumulation in the draining lymph nodes and the initiation of lymph node shut-down in response to a cutaneous West Nile Virus infection is dependent on interleukin-1beta and can occur in the absence of tumor necrosis factor.

Self-reported anxiety in children and adolescents: a three-year follow-up study.

Anxiety may be more transient in children and adolescents than in adults. The present study involves a longitudinal design enabling the investigation of the continuity/discontinuity of self-reported anxiety in children and adolescents. A sample of 68 children was followed over 3 years. Results indicate that, on the whole, self-reported anxiety decreased over time. This was true for overall anxiety and its sub-types, with the exception of social concerns/concentration, which did not decrease over time. Consistent with past research involving normal fear, girls and younger children were found to score higher on anxiety than boys and older children did. However, those groups scoring higher at inception also demonstrated the most marked decreases over the 3-year period. In addition to the changes found over time, the data indicated continuity in anxiety such that levels of anxiety at inception were significant predictors of follow-up anxiety, although only a small amount of variance was shared. The authors concluded that adult models of anxiety cannot be applied to youth and that future research should investigate the contribution of contextual factors to the development of anxiety in children.

School refusal in children and adolescents: a review of the past 10 years.

OBJECTIVE: To critically review the past 10 years of research on school refusal in children and adolescents. METHOD: Literature on school refusal published from 1990 onward was reviewed following a systematic search of PsycINFO. The review focuses on definitional issues, epidemiology and school refusal identification, diagnostic considerations, family functioning, assessment, treatment, and follow-up studies. RESULTS: While definitional and conceptual issues are still evident, promising developments have occurred in relation to assessment and treatment practices and understanding of the family context of school refusal. CONCLUSIONS: From a clinical viewpoint, school refusal cases require comprehensive assessment and treatment. Advances have been made in the treatment of school refusal. However, additional controlled studies evaluating interventions for school refusal are needed.

The intracellular oxidation of 2',7'-dichlorofluorescin in murine T lymphocytes.

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.

Children's nighttime fears: parent-child ratings of frequency, content, origins, coping behaviors and severity.

The present study investigated nighttime fears in normal school children aged 4 to 12 yr (N=176). Children and their parents were interviewed about the frequency, content, origins, coping behaviors and severity of children's nighttime fears. Results showed that 73.3% of the children reported nighttime fears, indicating that these fears are quite prevalent. Inspection of the developmental course of nighttime fears revealed that these fears are common among 4- to 6-year-olds, become even more frequent in 7- to 9-year-olds and then remain relatively stable in 10- to 12-year-olds. Inspection of the origins of nighttime fears revealed that most of the children (i.e., almost 80%) attributed their fear to negative information; conditioning and modeling were endorsed less frequently (25.6% and 13.2%, respectively). A substantial percentage of the children (24.0%) indicated that learning experiences had not played a role in the acquisition of their nighttime fears. Children reported a variety of coping strategies in order to deal with their nighttime fears and generally rated these strategies as helpful in reducing anxiety. Furthermore, children's nighttime fears were associated with moderate levels of anxiety. Moreover, in about 10% of the children, nighttime fears were related to one or more DSM-III-R anxiety disorders. Finally, parental reports of children's nighttime fears substantially deviated from children's reports. Most importantly, parents provided a marked underestimation of the frequency of nighttime fears, at least as reported by their children.

Treating sexually abused children with posttraumatic stress symptoms: a randomized clinical trial.

OBJECTIVE: To evaluate the efficacy of child and caregiver participation in the cognitive-behavioral treatment of sexually abused children with posttraumatic stress symptoms. METHOD: Thirty-six sexually abused children (aged 5-17 years) were randomly assigned to a child-alone cognitive-behavioral treatment condition, a family cognitive-behavioral treatment condition, or a waiting-list control condition. RESULTS: Compared with controls, children who received treatment exhibited significant improvements in posttraumatic stress disorder symptoms and self-reports of fear and anxiety. Significant improvements also occurred in relation to parent-completed measures and clinician ratings of global functioning. In general, parental involvement did not improve the efficacy of cognitive-behavioral therapy. Maintenance of improvement was evident at a 12-week follow-up assessment. CONCLUSIONS: Cognitive-behavioral treatment was useful, but further research is required on caregiver involvement.

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration.

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia.

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na(+) current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na(+) channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na(+) channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus.

Whereas there has been recent interest in interactions between dendritic cells and pathogenic viruses, the role of dendritic cells in the initiation of protective immunity to such organisms has not been elucidated. The aim of this study was to examine whether a resident dendritic cell population in the skin, Langerhans cells, respond to cutaneous viral infections which are effectively cleared by the immune system. We therefore characterized the ability of Langerhans cells to migrate to local draining lymph nodes following infection with the arthropod-borne viruses, West Nile virus or Semliki Forest virus. The data show that major histocompatibility complex class II+/NLDC145+/E-cadherin+ Langerhans cell numbers are increased in the draining lymph nodes of infected mice and this increase is accompanied by a concomitant decrease in the Langerhans cell density in the epidermis. Langerhans cell migration is associated with an accumulation of leukocytes in the lymph node, which is one of the earliest events in the initiation of an immune response. Both the migratory response and the draining lymph node leukocyte accumulation were abrogated if ultraviolet-inactivated instead of live viruses were used, suggesting the activation and subsequent migration of Langerhans cells requires a live, replicating antigen. Our findings are likely to have wider implications for the development of epidermally delivered vaccines and suggest that mobilization of dendritic cells may be involved in the development of immune responses to arthropod-borne viruses.

NADPH oxidase, Nramp1 and nitric oxide synthase 2 in the host antimicrobial response.

Using highly conserved, complex enzyme systems, leukocytes utilize the toxic nature of free radical intermediates, derived from oxygen and nitrogen, to control microbial pathogens as part of the innate immune response. Upon activation, NADPH oxidase generates superoxide anion radicals, which in turn give rise to further reactive oxygen intermediates. Similarly, activated nitric oxide synthase 2 catalyses the production of nitric oxide radicals, which leads to the formation of reactive nitrogen intermediates. Nitrogen- and oxygen-centered reactive intermediates can interact to form further reactive species. In addition, presence of the cationic transporter, Nrampl, may exacerbate the effects of these toxic compounds on invading microbes. While each of these antimicrobial systems can operate independently, the combination of their activities is synergistic in the successful containment of almost all invading pathogens. These systems are activated and modulated by microbial products and a series of temporally expressed cytokines. They also feed directly into the initiation of the adaptive immune response, which culminates in lasting specific immunity. The effector molecules, generated in the early innate immune response, are not specific to the invading pathogen and may also cause damage to the host. It is the critical balance of these processes in the initial stages of infection that determines the outcome of infectious disease.

Growth arrest-specific gene 6 expression in proliferating rabbit vascular smooth muscle cells in vitro and in vivo.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the processes of atherosclerosis and restenosis. The protein product of the growth arrest-specific gene 6 (Gas-6) has recently been identified as a ligand for the Axl/Rse/Mer tyrosine kinase receptor family, which may be involved in proliferation and migration of VSMCs. Here we show that Gas-6 gene expression is increased in proliferating VSMCs in tissue culture (2.5-fold increase by Northern blot) and following neointimal proliferation in a rabbit balloon-injury model (3-fold increase by Western blot). Neither platelet-derived growth factor (PDGF) nor thrombin stimulate the expression of Gas-6 in cultured VSMCs despite the ability of the PDGF, but not thrombin, to stimulate proliferation in growth-arrested cells. These data suggest a role for the Gas-6 regulatory system in VSMC proliferation, which may be a target for therapeutic interventions in the atherosclerotic process and restenosis after angioplasty.

Flow cytometry of plant cells with applications in large-scale bioprocessing.

In recent years, there has been a significant upsurge in the application of flow cytometry to plant cells and plant cell cultures. As well as a range of uses in plant biology, flow cytometry offers many advantages for monitoring plant cell cultures used in large-scale bioprocessing operations. This review summarizes the current status of the field, concentrating on methods for DNA measurement and multiparameter cell cycle analysis. Techniques for screening and selection of elite cell lines with high productivity of secondary metabolites are also addressed.

Empirically supported treatments for children with phobic and anxiety disorders: current status.

Reviews the empirically supported status of behavioral and cognitive-behavioral interventions in the treatment of childhood phobias and anxiety disorders. For childhood phobias, it is concluded that imaginal desensitization, in vivo desensitization, filmed modeling, live modeling, and cognitive-behavioral interventions that use self-instruction training are probably efficacious and that participant modeling and reinforced practice are well established. For anxiety disorders, only cognitive-behavioral procedures with and without family anxiety management (FAM) were found to be probably efficacious. However, much of the support for these procedures comes from analogue studies conducted in research laboratory or school settings, delivered in small-group format and, not infrequently, with nonclinically referred children. Additional research that examines high-strength interventions with clinic-referred children is recommended. Furthermore, research that examines the pathological processes involved in the onset and maintenance of phobic and anxiety disorders as well as the change processes used to treat these disorders is called for.