Biocide Exposure Induces Changes in Susceptibility, Pathogenicity, and Biofilm Formation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a frequent cause of catheter-associated urinary tract infection (CAUTI). Biocides have been incorporated into catheter coatings to inhibit bacterial colonization while, ideally, exhibiting low cytotoxicity and mitigating the selection of resistant bacterial populations. We compared the effects of long-term biocide exposure on susceptibility, biofilm formation, and relative pathogenicity in eight UPEC isolates. MICs, minimum bactericidal concentrations (MBCs), minimum biofilm eradication concentrations (MBECs), and antibiotic susceptibilities were determined before and after long-term exposure to triclosan, polyhexamethylene biguanide (PHMB), benzalkonium chloride (BAC), and silver nitrate. Biofilm formation was quantified using a crystal violet assay, and relative pathogenicity was assessed via a Galleria mellonella waxworm model. Cytotoxicity and the resulting biocompatibility index values were determined by use of an L929 murine fibroblast cell line. Biocide exposure resulted in multiple decreases in biocide susceptibility in planktonic and biofilm-associated UPEC. Triclosan exposure induced the largest frequency and magnitude of susceptibility decreases at the MIC, MBC, and MBEC, which correlated with an increase in biofilm biomass in all isolates. Induction of antibiotic cross-resistance occurred in 6/84 possible combinations of bacteria, biocide, and antibiotic. Relative pathogenicity significantly decreased after triclosan exposure (5/8 isolates), increased after silver nitrate exposure (2/8 isolates), and varied between isolates for PHMB and BAC. The biocompatibility index ranked the antiseptic potential as PHMB > triclosan > BAC > silver nitrate. Biocide exposure in UPEC may lead to reductions in biocide and antibiotic susceptibility, changes in biofilm formation, and alterations in relative pathogenicity. These data indicate the multiple consequences of biocide adaptation that should be considered when selecting an anti-infective catheter-coating agent.

Exposure to low-dose ultraviolet radiation suppresses delayed-type hypersensitivity to herpes simplex virus in mice.

Ultraviolet B (UVB) radiation is reported to induce a defect in epidermal antigen presentation which leads to specific suppression of the delayed-type hypersensitivity (DTH) response to trinitrochlorobenzene. We have used a similar system to examine the murine DTH response to herpes simplex virus type 1 (HSV-1). Mice irradiated with 96 mJ/cm2 UVB on shaved dorsal skin 3 days before s.c. injection of live HSV-1 in the flank showed 54-92% suppressed DTH responses to challenge with inactivated virus compared with nonirradiated control animals. If irradiation took place 7 days before inoculation with virus, some suppression of DTH occurred; if 14 days before, no suppression was found. The transient nature of the UVB response is further illustrated by the observation that irradiation with the same dose of UVB 5 h before, or 3 days after, inoculation with virus had no effect on DTH. Once induced, some degree of UVB suppression was found to persist for at least 3 months after irradiation.

Two phenotypically distinct T cells are involved in ultraviolet-irradiated urocanic acid-induced suppression of the efferent delayed-type hypersensitivity response to herpes simplex virus, type 1 in vivo.

When UVB-irradiated urocanic acid, the putative photoreceptor/mediator for UVB suppression, is administered to mice it induces a dose-dependent suppression of the delayed-type hypersensitivity response to herpes simplex virus, type 1 (HSV-1), of similar magnitude to that induced by UV irradiation of mice. In this study, the efferent suppression of delayed-type hypersensitivity by UV-irradiated urocanic acid is demonstrated to be due to 2 phenotypically distinct T cells, (Thy1+, L3T4-, Ly2+) and (Thy1+, L3T4+, Ly2-). The suppression is specific for HSV-1. This situation parallels the generation of 2 distinct T-suppressor cells for HSV-1 by UV irradiation of mice and provides further evidence for the involvement of urocanic acid in the generation of UVB suppression.

Ultraviolet-irradiated urocanic acid suppresses delayed-type hypersensitivity to herpes simplex virus in mice.

Ultraviolet radiation is known to induce a transient defect in epidermal antigen presentation which leads to the generation of antigen-specific suppression of the delayed-type hypersensitivity (DTH) response. The putative receptor in skin for the primary event in UV-suppression is urocanic acid (UCA) which may then interact locally, or systemically, with antigen presenting cells or initiate a cascade of events resulting in suppression. We present the first direct evidence that UCA, when irradiated with a dose (96 mJ/cm2) of UVB radiation known to suppress the DTH response to herpes simplex virus, type 1 (HSV-1) in mice, can induce suppression following epidermal application or s.c. injection of the irradiated substance. This suppression is transferable with nylon wool-passed spleen cells.

Interleukin-4 and interleukin-10 increase endotoxin-stimulated human umbilical vein endothelial cell interleukin-8 release.

The aim of this study was to determine the effect of interleukin-4 (IL-4) and interleukin-10 (IL-10) on interleukin-8 (IL-8) release from endothelial cells. Confluent monolayers of human umbilical vein endothelial cells (HUVECs) were incubated in the absence or presence of 10 ng/ml of bacterial lipopolysaccharide (LPS), with 5% human AB serum and recombinant human IL-4 or IL-10 over a dose range from 50 fg/ml to 50 ng/ml (final concentration). IL-4 and IL-10 had no effect on HUVEC IL-8 release in the absence of LPS. In the presence of LPS, IL-4 and IL-10 enhanced IL-8 release by approximately 300% compared with LPS-stimulated cells alone, IL-8 release increasing from 2594 +/- 493 pg/ml (no IL-4 or IL-10) to 7892 +/- 320 pg/ml (IL-4, 5 pg/ml; p = 0.001) and 8359 +/- 712 pg/ml (IL-10, 50 pg/ml; p = 0.002). IL-8 release in response to IL-4 or IL-10 plateaued above 5 and 50 pg/ml, respectively. This study suggests that IL-4 and IL-10 may be involved in the complex regulation of endothelial cell cytokine production during the response to endotoxin.

Murine HIV-1 p24 specific T lymphocyte activation by different antigen presenting cells: B lymphocytes from immunized mice present core protein to T cells.

Mice were injected with three doses of baculovirus-produced recombinant HIV-1 p24 core protein in alum adjuvant. CD4 positive T lymphocytes from immunized animals proliferated in vitro in the presence of antigen and peritoneal macrophages (Mps) or splenic dendritic cells (DCs) from non-immunized mice as antigen presenting cells (APCs). DCs were approximately three times more efficient than Mps on a cell for cell basis. No synergy was observed between Mps and DCs in this system. B lymphocytes from immunized animals also presented p24 antigen to the specific T cells. Mps did synergize with B cells to enhance the level of T lymphocyte proliferation. This may have implications for the induction of specific immune responses to pathogens after administration of single protein vaccines.

Induction of suppression of delayed type hypersensitivity to herpes simplex virus by epidermal cells exposed to UV-irradiated urocanic acid in vivo.

Urocanic acid (UCA), the putative photoreceptor for ultraviolet radiation (UV)-induced suppression, undergoes a UV-dependent trans to cis isomerisation. Epidermal cells from mice painted with UCA, containing a known proportion of the cis-isomer, generate suppression of the delayed type hypersensitivity response to herpes simplex virus type 1 (HSV-1) when transferred to naive syngeneic recipients at the same time and site as infection with HSV-1. One T suppressor cell subset, of phenotype (Thy1+, L3T4+, Ly2-), is induced by the cis-UCA modified epidermal cell transfer. Flow cytometric analysis of the epidermal cells from skin treated with UV or cis-UCA indicates an overall reduction from normal in the number of cells expressing MHC Class II antigens, but no alteration in the number expressing I-J antigens.

Lymphoproliferative and serological responses to herpes simplex virus during primary infection, recrudescence, and re-infection in a murine model.

Mice were infected epidermally with herpes simplex virus type 1 (HSV-1) after mild tape stripping. Some were re-infected by the same route several weeks later; recrudescences were induced in others by UV-irradiation before the primary infection followed by re-irradiation and tape stripping at a later date. Clinical symptoms were noted; serological responses to HSV and lymphoproliferative and phenotypic analysis of local lymph node cells were measured throughout. Experience of a primary lesion did not prevent lesions developing again on re-infection although morbidity and mortality were decreased. Recrudescent lesions were less severe than primary or secondary lesions, never zosteriform and healed rapidly. Antibodies to HSV were not found to play a major role in preventing development of lesions. The lymphoproliferative response on primary infection was maximal just after the lesions were most severe and then waned. There was a second, although not accelerated, lymphoproliferative response on re-infection with a persisting high level for at least one month. On recrudescence, limiting dilution culture analysis of lymphoproliferation demonstrated a recruitment within two days of HSV-1 specific lymphocytes to lymph nodes draining sites of lesions, which may limit their severity and duration.

Endogenous production of IL-8 by human colorectal cancer cells and its regulation by cytokines.

This study investigated the endogenous production of the pro-inflammatory and angiogenic cytokine IL-8 by human colorectal cancer cells. We describe substantial (>0.5 ng/ml) constitutive production of IL-8 by certain human colorectal cancer cell lines without the requirement for exogenous stimulation. Tumour cell-derived IL-8 production was upregulated by the addition of the pro-inflammatory type 1 cytokines TNF alpha, IL-1 beta and IFN gamma. Addition of the regulatory type 2 cytokines IL-4 and IL-10 produced paradoxical stimulation of IL-8 production in certain cell lines and downregulation of IL-8 production in others. These results suggest that tumour-derived cytokine production may have an important role in patients with colorectal cancer. Furthermore, they demonstrate the complexity of tumour cell cytokine production and highlight the difficulties in developing effective therapeutic biological response strategies.

Peripheral blood cells from weight-losing cancer patients control the hepatic acute phase response by a primarily interleukin-6 dependent mechanism.

Cancer cachexia is associated with an elevated hepatic acute phase protein response, poor outcome and elevated cytokine production from peripheral blood mononuclear cells (PBMC). This study investigates the mechanism by which PBMC can induce a hepatic acute phase response. Supernatants from the peripheral blood cells of cancer patients induced significantly higher C-reactive protein (CRP) from hepatocytes (198+/-21 ng ml-1) than did supernatants from healthy controls (64+/-20, p<0.005). CRP production in vitro correlated with IL-6 production by PBMC from patients with pancreatic cancer (r=0.76, p<0.0001). This C-reactive protein production was reduced by 84% using neutralising antibody to IL-6 (p<0.001). There was a significant negative correlation between PBMC-induced hepatocyte C-reactive protein production and survival (r=-0.45, p<0.01). PBMC from cancer patients induce the hepatic acute phase response via a primarily IL-6-dependent mechanism.

Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer.

Patients with pancreatic cancer frequently demonstrate symptoms such as weight-loss and muscle wasting and have clinical evidence of a systemic inflammatory response. Such effects may be mediated by pro-inflammatory cytokines derived from tumor cells. The production of interleukin-6 and -8 by pancreatic cancer cell lines and the influence of other cytokines on this production was studied. IL-8 was produced by all cell lines and production was increased following exposure to IL-1 and TNF. Cytokine-stimulated, but not basal IL-8 production was reduced by co-incubation with IL-4 in the MIA PaCa-2 and PANC-1 cell lines. The CFPAC cell line produced IL-6, but this production was not altered by IL-1, TNF or IL-4. In the PANC-1 cell line IL-8 and IL-8 receptors were only detected by PCR in cells which had been stimulated with TNF or IL-1. Serum concentrations of IL-6 and IL-8 were elevated in patients with pancreatic cancer compared with controls. In conclusion, human pancreatic cancer cell lines elaborate pro-inflammatory cytokines which have the potential to mediate elements of the systemic inflammatory response.

The effect of tamoxifen on tumours induced by cells from a canine mammary carcinoma cell line in athymic nude mice.

Tumours were induced in CBA athymic nude mice by subcutaneous injection of REM 134 cells. These cells were from a continuous line derived from a canine mammary carcinoma and have no detectable oestrogen receptors. In vitro, the anti-oestrogen tamoxifen was growth inhibitory at a concentration of 10(-6) M and adding 10(-8) M oestradiol-17 beta did not reverse this effect. The relative rate of growth of the tumours induced by the cultured cells was the same in male and female mice. Oral tamoxifen at a dose of 1 mg mouse-1 week-1 suppressed the early growth rate significantly and to the same extent in male and female mice; conversely subcutaneous tamoxifen at 2 mg mouse-1 week-1 had only a transient early effect. These results argue against tamoxifen acting solely as an antagonist for oestrogen.

Characteristics of a feline mammary carcinoma cell line.

The establishment of an epithelial cell line, JM, from a feline mammary adenocarcinoma is described. In vitro, the morphological and cultural properties of these cells, their surface features such as lectin binding and their response to hormones were ascertained. In vivo, they are tumorigenic in athymic nude mice inducing tumour nodules composed of epithelia-like cells.

Flexible training opportunities in the European Union.

This paper compares the opportunities for flexible (part-time) specialist training in the UK and elsewhere in the EU in the overall context of the rising numbers of women doctors across Europe. Few other EU countries appear to provide the same opportunities for flexible training as the UK, despite high percentages of women medical students and women medical graduates. There are important differences in training patterns across the EU and some reasons are proposed for why flexible training may be more difficult to implement or may not be required elsewhere in the EU. Reasons include less centralized health care systems and more rigidly structured training programmes. In the context of four main factors affecting medical manpower--medical unemployment, contracted working hours, maternity provisions and duration of training--both the health authorities' need to implement flexible training and the trainee doctors' demand for it would appear to be greater in the UK than in other EU countries.

Studies of three canine mammary cell lines--II. In vivo properties.

Three cell lines, REM 134, 111 and 367, derived from canine mammary carcinomas have been used to induce tumours in athymic nude mice after subcutaneous injection. The histopathology of the tumours was compared and each was found to resemble closely the original tumour. This did not change after serial in vivo passage. Metastasis never occurred. Injection of REM 134 cells intracranially resulted in a fast-growing tumour which also did not metastasize; injection intrapleurally resulted in growths most commonly on the mediastinum with confinement to the chest cavity. Fibronectin was present in the subcutaneous tumours. Two of the cell lines were cloned in semi-solid agar. When tested, these clones induced tumours identical histologically to the uncloned ones. Finally, male and female mice were injected subcutaneously with the same number of cells from each of the three lines but the rate of tumour growth did not differ significantly between the two sexes.

The immune response to glycoproteins of herpes simplex virus.

Herpes simplex virus commonly causes 'cold sores', the virus often becoming latent after the primary infection and recrudescing at intervals. Six glycoproteins have been described in the virion and in the membranes of infected cells; much of the host's immune response is directed against these molecules. The functions of individual glycoproteins in the disease process and in the induction of different components of the immune response are beginning to be unravelled, which should help provide a strategy for the creation of an effective subunit vaccine against the virus.

Interactions between herpes simplex virus and murine bone marrow macrophages.

Macrophages from murine bone marrow (strain C3Hf Bu/Kam) were cultured in vitro in L-cell conditioned medium. After 0, 2, 4, 6, 8 and 10 days, they were infected with a clinical strain of herpes simplex virus type 1 and the outcome followed morphologically, by phagocytic index, infectious virus yields, immunofluorescence, expression of Fc receptors and major histocompatibility complex (MHC) Class II antigens. At a multiplicity of infection of 1-5, little morphological difference was apparent between infected and uninfected cultures at early stages in vitro but marked changes occurred later with reduction in cell numbers in the infected cultures. Indirect immunofluorescence failed to detect cells expressing early viral antigens, and yields of infectious virus indicated that permissive infection was not taking place. While phagocytic index and Fc receptor expression did not change 24 hours post-infection, MHC Class II antigen expression was increased. Thus, although the bone marrow macrophages seem predominantly resistant to infection with HSV-1, they may be modified by the presence of the virus. Since macrophages may act as antigen presenting cells for the immune system, this type of mechanism may be important in the generation of local immune responses.

The effect of UV-irradiation on viral infections of the skin.

The effect on the skin of exposure to relatively small doses of ultraviolet light has long been thought beneficial and socially desirable, leading to a 'healthy tan'. However it is less well known that similar (if not smaller) doses of UV-B radiation can have transient but profound depressive effects on the immune response to antigens or pathogens encountered by the skin. The result of this on the immunopathology of both primary and recurrent episodes of persistent viral infections of the epidermis is discussed.

Alterations in epidermal handling of HSV-1 antigens in vitro induced by in vivo exposure to UV-B light.

In order to investigate the cellular interactions involved in the immune response to herpes simplex virus type 1 (HSV-1) in a murine model, an in vitro antibody induction system was developed. This comprised HSV-1-primed T cells from infected mice, trinitrophenol (TNP)-primed B cells from mice primed with TNP-coupled calf erythrocytes, and TNP-HSV-1 as antigen. When antigen-presenting cells (APC) were removed from the assay system, the induced antibody response disappeared but could be reconstituted by the addition of APC derived from the peritoneal cavity or skin of normal mice. Since HSV-1 is an epidermal pathogen, it was decided to investigate the role of skin APC in HSV-1 immunity. Skin APC from mice irradiated 3 days previously with a suberythemal dose of ultraviolet (UV)-B were found to have a decreased capacity to present HSV-1 antigen in vitro. However, the APC capacity of their peritoneal cells was unaffected. The reduction in APC capacity is not only a local effect at the irradiated site, as APC from mice exposed to UV-B with one ear protected by black electrical tape were equally affected in both ears.

Studies of three canine mammary carcinoma cell lines--I. In vitro properties.

Three cells lines, REM 134, 111 and 367, have been derived from canine mammary carcinomas and their morphological characteristics in vitro are described. They are tumorigenic in athymic nude mice, have no demonstrable fibronectin on their cell surfaces and exhibit a varied pattern of lectin binding. They can be cloned in semi-solid agar. One line, REM 134, responds to oestrogen and luteotropic hormone in vitro, although none of the three had demonstrable oestrogen receptors.