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.

1H-NMR visible neutral lipids in activated T lymphocytes: relationship to phosphatidylcholine cycling.

Two-dimensional 1H-NMR spectroscopy was used to compare changes in the concentration of isotropically-tumbling neutral lipid during the activation of splenic and thymic T lymphocytes. The concentration of mobile neutral lipid (MNL) was similar in splenic and thymic T cells after 72 h of activation with phorbol myristate acetate and ionomycin. However, after 120 h of activation, MNL concentrations in splenic T cells were more than 3-fold higher than in thymic T cells. An increase in choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) was also observed in both thymic and splenic T cells after 24 h of activation. However, after 72 h of stimulation, Cho and PCho levels had decreased and continued to decline at 96-120 h, while GPC continued to be maintained at elevated levels. The simultaneous increase in MNL and GPC and the decline in Cho and PCho leads us to propose that the synthesis of NMR-visible MNL in activated lymphocytes is linked to the phosphatidylcholine cycle.

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.

The surface concentration of H-2 antigens on mouse pancreatic beta-cells and islet cell transplantation.

Measurement of the concentration of H-2 complex antigens in the surface membrane of mouse pancreatic beta-cells by a quantitative immunoferritin-labeling technique revealed that the antigens were present in very small amounts on the beta-cells of five strains. A comparison of the labeling densities in the five strains suggested that beta-cells from C57BL/10Sn congenic strains have about half the H-2 antigen density of BALB/c and C3H/HeJ cells. In C57BL/10Sn mice the antigen density on beta-cells was slightly greater than that on erythrocytes, about 20% of that on thymocytes, and about 2.5% of that present on peritoneal macrophages. Intraperitoneal allografts of c57BL/10Sn islets were uniformly rejected by B10.BR/SgSn diabetic recipients only when the islets were accompanied by 10(7) peritoneal lymphoid cells. When transplanted without peritoneal cells, C57BL/10Sn islets were only marginally rejectable. In a group of nine such allografts, three diabetic recipients were permanently cured and three others showed rejection times of about 30 days. Sensitization of the three mice showing permanent cures, using 10(7) allogeneic peritoneal cells at about 40 days after the transplant, did not cause rejection of the allografts. Isogeneic transplantation of cell suspensions from dissociated islets of Langerhans was markedly less effective in controlling diabetes than intact islets, and dissociation did not obviously improve the rate of allograft survival. However, 5/19 diabetic mice receiving allografts of dissociated islet cells showed long-term reversals of diabetes that were unaffected by administration of 10(7) peritoneal cells at about 100 days after the transplant. Recipient mice whose diabetes was reversed by islet allografts and unaffected by specific sensitization had pancreatic insulin concentrations characteristic of diabetic mice. Our reversals of diabetes with untreated islet allografts may be due to the cleanliness of islet preparations obtained with a modified isolation technique, and to the very low density of H-2 complex antigens on C57BL/10Sn beta-cells.

HIV infection of placental macrophages: their potential role in vertical transmission.

Placental macrophages were isolated and cultured in vitro to investigate their susceptibility to HIV infection and possible role in vertical transmission of HIV. After 10 days of in vitro culture the cells were positive for nonspecific esterase and acid phosphatase and negative for myeloperoxidase and placental alkaline phosphatase. They expressed cell surface HLA-ABC, HLA-DR, CD45, as well as CD68 intracellularly, as detected by flow cytometry, confirming their macrophage lineage. Approximately 80% of cells expressed surface CD14. CD4 antigen was expressed at very low levels and was confirmed by antibody blocking experiments. Infection of placental macrophage cultures with HIV resulted in a transient peak of viral replication 3 to 7 days after infection, but no later rise in HIV was detected with culture of up to 60 days. HIV replication was not up-regulated by coculture with phytohemagglutinin-stimulated lymphocytes or by treating infected cultures with tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor.

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.

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.

Magnetic resonance studies of murine macrophages. Proliferation is not a prerequisite for acquisition of an 'activated' high resolution spectrum.

Proton magnetic resonance spectroscopy (1H-MRS) was used to investigate the membranes of macrophages activated by gamma-interferon in vitro and by Listeria monocytogenes in vivo. We report the appearance with activation, of a high resolution spectrum indistinguishable from that found in activated T and B cells and embryonic and malignant cell types previously studied. We furthermore show that proliferation is not a prerequisite for the appearance of this activated spectrum. This supports the idea that membrane 'activation' in all cells, irrespective of origin, may be accompanied by similar architectural changes, and suggests that a common pathway exists for the activation of cell membranes of the immune system, possibly important in the acquisition of increased motility. The use of 1H-MRS as a non-invasive tool for analysis of activation is discussed.

Na(+)-dependent high affinity uptake of L-glutamate in primary cultures of human fibroblasts isolated from three different types of tissue.

Cultured human fibroblasts isolated from embryonic muscle, skin and peripheral nerve tissues were found to accumulate [3H]L-glutamate by a Na(+)-dependent uptake process strongly inhibited by several glutamate/aspartate analogues including D- and L-aspartate, D- and L-threo-3-hydroxyaspartate and L-trans-pyrrolidine-2,4-dicarboxylate but not D-glutamate. It was also reduced by elevated concentrations of K+, Rb+ and Cs+. The values of Km's were 5-20 microM, well within the 'high affinity' region. Variations in the capacity (Vmax) of [3H]L-glutamate uptake did not correlate with the origin (muscle, skin or nerve tissue) of the fibroblasts. The uptake characteristics suggest that it is mediated by a transport system similar to that commonly observed only in brain tissue.

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.

Immuno-gold labeling for flow cytometric analysis.

Colloidal gold particles coated with goat anti-mouse immunoglobulin antibodies were used to analyse surface antigens on various cell types by flow cytometry. The gold-labeled cells showed an increasing signal amplification in the 90 degree light scatter with increasing wavelength of the incident laser light, reaching a more than 10-fold amplification at 632.8 nm. This wavelength was provided by a 0.5 mW helium-neon laser. The magnitude of the signal amplification due to the gold label as well as the specificity of the label was sufficient for quantitative discrimination between positive and negative cells. Cell viability was not affected by the gold label. Mouse spleen cells were labeled with various combinations of FITC- and gold-conjugated antibodies. It was found that the gold and fluorescent labels did not interfere with each other. Colloidal gold may thus be used as an additional label for multiparameter cell analysis and sorting. Biparametric cell analysis/sorting of surface antigen-labeled cells (label versus low-angle scatter) becomes possible even with a low energy helium-neon laser.

Ontogeny of major histocompatibility complex antigen expression on cultured human embryonic skeletal myoblasts.

Human embryonic myoblasts isolated from 13- to 19-week embryos were treated for 24 to 144 hr with 0.1-500 U/ml IFN-gamma and the constitutive and IFN-gamma-inducible MHC expression was examined by flow cytometry. Low levels of constitutive MHC I were expressed that increased with both developmental age and incubation time. In contrast, no constitutive MHC II was detected on human embryonic myoblasts at any age or incubation time. Both classes of MHC can be induced by IFN-gamma. Maximal MHC I induction increased in parallel with age, i.e., maximal induction occurred on 19-week myoblasts, while MHC II induction peaked at 17 weeks. IFN-gamma-induced expression of MHC I and II also increased with incubation time. Induced expression of MHC I antigen reached plateau levels at 72 hr of IFN-gamma incubation, whereas MHC II increased to a plateau level at 120 hr. The immunological importance of these findings for myoblast transfer therapy is discussed.

The effects of West Nile virus on major histocompatibility complex class I and II molecule expression by Lewis rat Schwann cells in vitro.

The expression of major histocompatibility complex (MHC) class I and II molecules by Lewis rat Schwann cells after infection with West Nile virus (WNV) in vitro was examined by fluorescence microscopy and flow cytometry. WNV enhanced the expression of MHC class I molecules and induced the expression of MHC class II molecules by Schwann cells. Irradiated medium from WNV-infected Schwann cell cultures upregulated class I molecule expression on dissociated Schwann cell cultures but did not induce the expression of class II molecules. This finding has implications for virally triggered autoimmune diseases of nervous tissue.

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.

West Nile virus infection induces susceptibility of in vitro outgrown murine blastocysts to specific lysis by paternally directed allo-immune and virus-immune cytotoxic T cells.

Day 3 post-coitum BALB/c and (BALB/c x CBA/H)F1 blastocysts were isolated and hatched in replicate wells. Some were treated with interferon-gamma (IFN-gamma). Whilst others were infected with West Nile Virus (WNV) at 100 plaque-forming units per cell, for 18 h. Controls were mock-treated. Gamma-irradiated (2000 rads) CBA/H, (paternal) WNV-specific and allo(CBA/H)-specific cytotoxic T (Tc) cells were then added to replicates of infected, mock-infected or IFN-gamma-treated cultures for 20 h. [3H]Thymidine was then added for a further 8 h. [3H]Thymidine incorporation was inhibited by 40-50% in WNV-infected cultures exposed to WNV-paternal-specific Tc cells and by 30-40% in WNV-infected cultures exposed to allo-paternal-specific Tc cells compared to similarly exposed, uninfected, or unexposed, WNV-infected, or unexposed, uninfected cultures. No significant differences in [3H]thymidine incorporation were found between these controls and IFN-gamma-treated cultures exposed to allo-paternal-specific Tc cells or IFN-gamma-treated cultures not exposed to Tc cells. Parallel exposure of L929 fibroblasts to the same Tc cells irradiated with 500-8000 rads in doubling doses, showed that irradiation did not alter the efficacy or specificity of the Tc cells. Relevance to maternal anti-viral immune responses during implantation is discussed.

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro. II. The role of gamma interferon in the responses of primary and secondary giant cells.

The induction of paternal Class I and II MHC antigens by crude lymphokine preparations or purified recombinant gamma interferon was investigated on (C57BL/6J X CBA/H)F1 primary and secondary trophoblast giant cell outgrowths from 3.5-day post-coital (pc) blastocyst and 7.5-day pc ectoplacental cone preparations, respectively, using sensitive immunogold labelling techniques and electron microscopy. Class I MHC (but not Class II) antigens could readily be induced on secondary trophoblast giant cells, by incubation in vitro with gamma interferon for 40 h. However, repeated attempts to induce detectable MHC antigens on primary trophoblast giant cells failed. Mock-treated (C57BL/6J X CBA/H)F1 secondary trophoblast giant cell control preparations failed to express detectable MHC antigens. These findings suggest that, at the time of implantation, there is a time window during which MHC antigens are neither expressed constitutively nor are inducible by soluble factors which normally modulate cell surface MHC antigen concentration.

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro.

The expression of paternal class I and class II major histocompatibility complex (MHC) antigens in cultures of murine ectoplacental cone trophoblast was examined using immunogold labelled antibodies and electron microscopy. Class I MHC antigens could be induced on ectoplacental cone derived trophoblast following exposure to concanavalin A stimulated T cell supernatants. Class I MHC antigens were not detected in untreated trophoblast cultures. Class II MHC antigens were never detected on trophoblast whether treated or untreated. This is the first report of the experimental induction of Class I MHC antigens on a population of normally MHC-negative trophoblast cells.

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.

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.