Rapid identification of uropathogenic Escherichia coli of the O25:H4-ST131 clonal lineage using the DiversiLab repetitive sequence-based PCR system.

Several recent studies have highlighted the emergence of a globally disseminated clone of uropathogenic and invasive Escherichia coli isolates of serotype O25:H4 and sequence type 131. The ability to characterize rapidly E. coli isolates of this lineage would facilitate enhanced surveillance for this pathogen. We have used the semi-automated DiversiLab repetitive PCR-based system to analyse a collection of 35 clinical isolates of uropathogenic E. coli from across the UK, with particular focus on the O25:H4-ST131 lineage. All isolates had been characterized using multilocus sequence typing (MLST), and 14 had previously been typed using pulsed-field gel electrophoresis (PFGE). The DiversiLab system allowed discrimination of O25:H4-ST131 isolates from those of other E. coli lineages. It was slightly more discriminatory than MLST, but was less discriminatory than PFGE. With an analysis time of <4 h between receipt of a cultured organism and provision of a typing result, the system offers information on a real-time basis, a major advantage over current practice. We suggest that introduction of the DiversiLab system would be useful for rapid exclusion of E. coli isolates during outbreak investigations, and that the approach could be employed for surveillance for pathogenic or antibiotic-resistant clones of this organism.

Interleukin-6 and tumor necrosis factor-alpha levels following hepatic cryotherapy: association with volume and duration of freezing.

Although morbidity following cryotherapy is usually minor, a syndrome of multiorgan failure and disseminated intravascular coagulation (DIC) has been described and referred to as the cryoshock phenomenon. We hypothesized that mediators similar to those in septic shock may be involved in this syndrome. In this study we aimed to assess the plasma concentrations of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) following hepatic cryotherapy and to relate them to the duration and volume of freezing and to hepatocellular injury. Between April and December 1997 blood samples were taken preoperatively and at different times postoperatively from patients undergoing hepatic artery catheter-insertion (HAC) (n = 15), cryotherapy (n = 5), liver resection (n = 9), liver resection and edge cryotherapy (n = 7), or liver resection and cryotherapy of additional lesions (n = 9). They were analyzed for serum aspartate transaminase (AST) and plasma TNF-alpha and IL-6 levels. There was a significant association (Pearson correlation) of serum AST levels 1 hour postoperatively with plasma TNF-alpha and IL-6 levels at the end of the procedure. In patients undergoing cryotherapy or resection with cryotherapy of additional lesions (n = 14), the volume and duration of hepatic freezing were significantly associated with postoperative serum AST and plasma TNF-alpha and IL-6 levels at various postoperative times. Hepatic cryotherapy is followed by cytokine release, with postoperative plasma TNF-alpha and IL-6 levels associated with the degree of hepatic cryotrauma. These mediators may be involved in the occurrence of cryoshock following large-volume hepatic freezing.

Study on the reduction of chemotherapy induced neutropenia in mice using glucosaminylmuramyl dipeptide.

Neutropenia is a common and often dose limiting side effect of some chemotherapy regimens. The aim of this study was to investigate the ability of an immunomodulator, glycosaminylmuramyl dipeptide (GMDP, CAS 78113-36-7, romurtide) to reduce chemotherapy induced neutropenia. BALB/c mice were treated with 200 mg kg-1 cyclophosphamide (CY) to induce a reversible neutropenia lasting approximately 6-7 days. There was no change in the duration or depth of neutropenia in mice treated with GMDP for 3 consecutive days (2.5 or 25 mg kg-1) starting the day after CY injection. In addition, at the doses used, the time of administration of GMDP relative to CY did not alter this response. However, a marked neutrophilia compared to controls was consistently observed in all cases. Neutrophil counts of up to 14 times the baseline value were measured 6-7 days after the induction of neutropenia. GMDP had no effect in the absence of CY. Less variation was seen in white cell counts of older non-SPF mice treated with CY. When the activity of GMDP (5 mg kg-1) was compared with G-CSF (granulocyte colony stimulating factor, 100 micrograms kg-1) in these animals, GMDP showed a consistent trend to reduce the length of neutropenia, however, under the conditions tested only G-CSF treatment resulted in a significant reduction in the duration of neutropenia. In the 12-week-old mice, the neutrophilia seen with both G-CSF and GMDP was much smaller than in the 8-week-old mice, and was not significantly different from that in control mice treated with CY alone.

Loading with oxidised low density lipoprotein alters endocytic and secretory activities of murine macrophages.

Lipid-loaded macrophages were produced in vitro by incubation with acetylated or copper-oxidized LDL. In order to establish whether cellular membrane traffic is generally perturbed by such loading, we assessed endocytosis of fluid; cell surface binding, internalisation and degradation of a soluble ligand and of a particulate preparation; and exocytosis of lysosmal enzymes. Fluid-phase pinocytosis of sucrose was unaffected by either form of loading. Binding, uptake and degradation of soluble (mannosylated-BSA) and particulate (zymosan) ligands by these lipid-loaded and by non-loaded cells were compared. Loading with oxidized LDL decreased the processing of both ligands, while loading with acetylated LDL had little effect. Loading with oxidized LDL (Ox-LDL) also decreased zymosan binding at 4 degrees C; and the internalisation and degradation of ligands in Ox-LDL loaded and non-loaded cells reflected the extent of surface binding. Changes in binding and uptake of mannosylated-BSA and zymosan were not due to changes in viability or cell number. Zymosan stimulated release of lysosomal beta-N-acetyl-D-glucosaminidase from the cells. Loading with Ox- but not Ac-LDL decreased beta-N-acetyl-D-glucosaminidase secretion. After incubation with zymosan, intracellular levels of the enzyme were increased in the Ox-LDL loaded cells. Zymosan uptake and beta-N-acetyl-D-glucosaminidase secretion were correlated, but enzyme activity per culture rose more in the absence than in the presence of zymosan. We conclude that membrane traffic is perturbed in model foam cells, particularly those loaded with Ox-LDL.

Vitamin D3 analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model.

PURPOSE: In this study, we investigated the effect of the vitamin D3 analog, EB1089, on the growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model. METHODS: BALB/c Nu/Nu nude mice were inoculated subcutaneously with 10(6) LoVo cells. EB1089 dissolved in isopropanol was administered intraperitoneally and orally on alternate days at doses of 0.1, 0.5, and 2.5 microg/kg/day. Control animals received isopropanol alone. Tumor volumes estimated using the formula 0.5 X length X (width)2. The tumor kinetic index was determined by immunohistochemical detection of proliferating cell nuclear antigen. RESULTS: Significant dose-dependent inhibition of tumor growth was seen. After 20 days of treatment with 0.1 microg/kg/day EB1089, mean tumor volume in treated mice was 41 to 49 percent less than that in control animals (P < 0.01). Significant inhibition of tumor growth was also seen with 0.5 microg/kg/day EB1089 after 22 days of treatment (51 percent of control P < 0.01). Treatment with 2.5 microg/kg/day resulted in weight loss that required termination of this group; these mice were subsequently found to be hypercalcemic. The tumor kinetic index was significantly lower in tumors treated with 0.1 microg/kg/day EB1089 compared with that for control tumors (8 vs. 30 percent in controls). CONCLUSION: These findings suggest that the vitamin D3 analog, EB1089, is a potent antiproliferative agent for some human colon cancers.

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide.

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper-oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFN gamma and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68-99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFN gamma/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFN gamma/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Immunoregulation in the common marmoset, Calithrix jaccus: functional properties of T and B lymphocytes and their response to human interleukins 2 and 4.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.

Functional and phenotypic analysis of human T-cell clones which stimulate IgE production in vitro.

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29.