Artificial lighting reduces the effectiveness of wildlife-crossing structures for insectivorous bats.

In an attempt to improve cost-effectiveness, it has become increasingly popular to adapt wildlife crossing structures to enable people to also use them for safe passage across roads. However, the required needs of humans and wildlife may conflict, resulting in a structure that does not actually provide the perceived improvement in cost-effectiveness, but instead a reduction in conservation benefits. For example, lighting within crossing structures for human safety at night may reduce use of the structure by nocturnal wildlife, thus contributing to barrier and mortality effects of roads rather than mitigating them. In this study, we experimentally evaluated the impact of artificial light at night on the rate of use of wildlife crossing structures, specifically underpasses, by ten insectivorous bat species groups in south-eastern Australia. We monitored bat activity before, during and after artificially lighting the underpasses. We found that bats tended to avoided lit underpasses, and only one species consistently showed attraction to the light. Artificial light at night in underpasses hypothetically increases the vulnerability of bats to road-mortality or to the barrier effect of roads. The most likely outcomes of lighting underpasses were 1. an increase in crossing rate above the freeway and a decrease under the underpasses, or 2. a reduction in crossing rate both above freeways and under the underpasses, when structures were lit. Our results corroborate those of studies on terrestrial mammals, and thus we recommend that underpasses intended to facilitate the movement of wildlife across roads should not be lit.

PO-21 - Stromal fibroblasts in preinvasive breast cancer (ductal carcinoma in situ, DCIS) demonstrate a cancer-like procoagulant phenotypic switch that may facilitate invasion.

INTRODUCTION: Ductal carcinoma in-situ (DCIS) is a preinvasive breast cancer where cancer cells remain confined within the ductal basement membrane. However, genotypic changes have been identified in stroma surrounding DCIS, outside the basement membrane. Stromal fibroblasts undergo phenotypic change in cancer to promote tumour angiogenesis, proliferation, immunosuppression and metastasis and in vivo can induce invasion of DCIS. Phenotypic changes in DCIS stromal fibroblasts may potentially act as a precursor for invasion. AIM: To determine if stromal fibroblasts in DCIS have procoagulant changes similar to those seen in cancer-associated fibroblasts in invasive breast cancer. MATERIALS AND METHODS: As part of the prospective cohort study CHAMPion (Cancer induced Hypercoagulabulity as a Marker of Prognosis), patients with DCIS (n=72) and invasive breast cancer (n=292) were recruited. Stromal fibroblasts in tumour and corresponding normal breast tissue (distant from the cancer) were quantified (percentage IHC stained) for tissue factor (TF), thrombin, PAR1 and PAR2. Fibroblasts were identified morphologically, at a minimum distance of 0.2mm from ductal tissue, to avoid myoepithelial scoring. Scoring was performed in duplicate by two independent pathologists. RESULTS: Fibroblast TF expression was present in normal breast tissue (mean 43% ([SD 27%]) but markedly increased in DCIS (mean 62% [SD 27%], p=0.002). Fibroblast TF expression was further increased in invasive breast cancer (mean 74% [SD 23%], normal vs invasion, p<0.001; DCIS vs invasion, p=0.03). Fibroblast thrombin and PAR2, but not PAR1, expression was increased in DCIS compared to normal (thrombin: 60% vs 42%, p<0.001; PAR2: 58% vs 41%, p=0.002), however no further significant increase was seen in invasive cancer (thrombin 63%, PAR2 61%). Fibroblast tissue factor correlated with fibroblast thrombin expression (p<0.001, r=0.4) and fibroblast PAR2 expression (p<0.001, r=0.5), with thrombin and PAR2 expression also correlating (p<0.001, r=0.4). CONCLUSIONS: Procoagulant phenotypic changes, in terms of increased TF, thrombin and PAR2 expression, occur in stromal fibroblasts at the preinvasive stage. It needs to be determined if this change is functional and therefore a potential therapeutic target for preventing transition to invasion.

Choroidal dendritic cells require activation to present antigen and resident choroidal macrophages potentiate this response.

BACKGROUND/AIM: The uveal compartment of the eye contains extensive networks of resident macrophages and dendritic cells. These cells are now recognised to have a role in many ocular pathologies. The aim of this study was to isolate, characterise, and compare the function of ciliary body/choroid dendritic cells and macrophages from the normal eye. METHODS: Explants of rat and human ciliary body/choroid were cultured in vitro for various periods of time and cells harvested either from the supernatant fluid or from enzyme digested and washed explants. The cells were then phenotyped by microscopy and flow cytometry, examined by video time lapse photomicroscopy, and analysed functionally in a series of immunoassays. RESULTS: Two main types of dendritic cell were identified: large veil-like MHC class II(mid) motile but relatively non-translocatory cells and small MHC class II(hi) motile and rapidly translocating cells. Tissue macrophages mainly remained associated with the explants in culture but gradually lost their resident tissue marker (ED2) and detached from the explants as clusters of low density, large, CR3 (ED7)(+) cells, some of which underwent apoptosis. Video time lapse studies showed dendritic cells constantly interacting with large single cells and cell clusters by traversing the interstices of the cell clusters. In functional studies, freshly isolated dendritic cells were poor presenters of antigen and required activation by short term culture for acquisition of antigen presenting function. In contrast, dendritic cell depleted choroidal cell preparations containing macrophages and other cells failed to present antigen even after short term culture but augmented the antigen presenting function of dendritic cells when tested in co-culture. CONCLUSION: At least two types of dendritic cells are present in the normal ciliary body/choroid layer of the eye. It is likely that these cells have different functions based on their motility and potential to migrate to secondary lymphoid tissue either during normal physiological homeostatic processes or during an inflammatory response. The behaviour of resident tissue myeloid cells may decide the outcome of the organism's response to stress, foreign antigen, and ageing processes such as age related macular degeneration.

Kinetics of leukocyte and myeloid cell traffic in the murine corneal allograft response.

BACKGROUND: Little information exists on the trafficking of myeloid and lymphoid cells between the transplanted cornea and the secondary lymphoid tissue. This study reports on changes in the cornea and the draining lymph node (DLN) from the time of graft emplacement. METHODS: Using a mouse corneal graft model (C57BL/10Sn to BALB/c), eyes and submandibular DLN were examined by immunohistochemistry and three-color flow cytometry for evidence of T cell activation and dendritic cell (DC) conditioning (up-regulation of costimulatory molecules) at various times (15 min to 24 days; n=4 for each time). RESULTS: In the DLN, early (2 hr) DC conditioning was sustained throughout allograft rejection whereas a remarkable drop in percentage of activated CD4+ and CD8+ T cells (P <0.001) was followed by a biphasic rise in activated CD4+ and, to a lesser extent, CD8+ T cells (24 hr, P <0.001 and 6 days, P <0.01). CD11b+ and MOMA-2+ macrophages, MHC Class II+ cells, CD86+ DC, and neutrophils were the earliest cells infiltrating the cornea (at 24 hr), whereas T cells appeared after 2 days, with CD4+ T cells being confined largely to the graft recipient border. CONCLUSIONS: Immediate and rapid changes in T cell and DC populations in the DLN correlate with the type of cellular infiltration in the corneal graft. The data are consistent with a model in which CD4+ T cell help for CD8+ cytotoxic T cells could be provided by sequential two-way activation of T cells and DC in the DLN. The majority of cells infiltrating the graft were macrophages and neutrophils, with fewer DC and T cells.

IL-18 not required for IRBP peptide-induced EAU: studies in gene-deficient mice.

PURPOSE: Interleukin (IL)-18 has been described as a proinflammatory cytokine in rheumatoid arthritis and bacterial infectious diseases. The present study was designed to determine the role of IL-18 in a model of ocular experimental autoimmune uveitis (EAU). The initial studies were conducted to detect the expression of IL-18 in normal mouse eye tissue, and the later studies investigated induction of EAU in mice with an IL-18(-/-) phenotype. METHODS: IL-18 detection was performed by using 5-bromo-4-chloro-3-indoyl-ss--D-galactopyranoside (X-Gal) staining on frozen sections of eyes from mice (129/CD1, DBA1, and Balb/c), either of normal phenotype (+/+) or of deficiency (+/-, -/-) in the IL-18 gene which had been replaced by introduced genes including LacZ under the control of an IL-18 promotor. Severity of EAU was assessed in DBA1 and 129/CD1 wild-type (WT) or IL-18 knockout (KO) mice after immunization with the uveitogenic antigen: interphotoreceptor retinal binding protein (IRBP) peptide 161-180. Lymphocyte proliferation and cytokine production were also measured in WT and IL-18 KO DBA1 mice 15 days after immunization. RESULTS: IL-18 is constitutively expressed in the epithelial cells in iris, ciliary body, and retina. EAU-resistant mice (129/CD1) with an IL-18(-/-) phenotype remained resistant after immunization with IRBP peptide (P161-180). However, EAU-susceptible mice (DBA1) exhibited disease with similar histologic characteristics, despite a generalized reduction of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on an IL-18(-/-) phenotype. DBA1 IL-18(-/-) also demonstrated reduced IL-10 production. CONCLUSIONS: The IL-18 gene is not necessary for the initiation or pathogenesis of EAU induced by IRBP peptide 161-180. IL-18 is expressed in the epithelial cells in iris, ciliary body, and retina in the eyes, but its role in the eye remains undetermined.

Macrophages and dendritic cells in IRBP-induced experimental autoimmune uveoretinitis in B10RIII mice.

PURPOSE: To investigate the characteristics of the mononuclear cell infiltrate in murine experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced by immunization with bovine interphotoreceptor retinal binding protein (IRBP) in Freund's complete adjuvant (subcutaneous injection) and pertussis toxin (intraperitoneal injection) in B10RIII mouse. Then animals were killed on days 7, 9, 12, 15, 20, 26, and 39 after immunization. Eyes were processed for hematoxylin and eosin staining to characterize the disease and to assess the severity and extent of the EAU. Single and dual immunohistochemical staining in various combinations with monoclonal antibodies against CD45, CD4, CD8, major histocompatibility complex (MHC) class II, CD11c, NLDC-145, and a variety of macrophage markers was performed. RESULTS: The authors' results showed that vitritis, vasculitis and perivasculitis, retinal detachment, and granuloma formation in retina and choroid were the predominant features of IRBP-induced B10RIII mice EAU. Immunohistologic results showed that CD4+ T cells and macrophages were the main infiltrating cells in retina and choroid throughout the entire course of the disease. MHC class II negative macrophages expressing antigens reacting with MOMA-2, F4/80, sialoadhesin, and CD11b were prominent during the peak phase of tissue damage in the retina and choroid. Dendritic cells (DCs) characterized by dual positivity for MHC class II and CD11c and negative for sialoadhesin appeared at time of disease onset and continued to be recruited during the inflammatory process. DCs at the site of inflammation were NLDC-145 weak and CD8 negative, indicating that they were of the myeloid rather than the lymphoid lineage. CONCLUSIONS: The results suggest that EAU in B10RIII mice is initiated by local-infiltrating, dendritic antigen-presenting cells, whereas tissue damage is associated with sialoadhesin-positive, phagocytic nonantigen-presenting macrophages during the effector stage.

Cell subpopulations in failed human corneal grafts.

BACKGROUND/AIMS: Inflammatory cells and antigen presenting cells (APC) are not present under normal circumstances in the centre of the healthy cornea. The purpose of this study was to investigate and phenotype the inflammatory cell populations, particularly with reference to T cell subpopulations and macrophages, and to localise dendritic cells (DC) and other MHC class II positive cells in three groups of grafted corneas: rejected non-inflamed, rejected inflamed grafts, and control dystrophic explants. METHODS: 15 corneal buttons removed during keratoplasty from non-inflamed "quiet" previously grafted corneas, five inflamed corneas requiring urgent regrafting for "graft melting" (in "high risk" corneas), and 10 control dystrophic opaque corneas explanted during their first graft procedure were examined. Cryosections of corneas were immunostained with a panel of monoclonal antibodies (mAb) against CD3, CD4, CD8, CD14, CD25, CD68, HLA-DP, and HLA-DR molecules using the StreptABC method. DC were detected by dual immunostaining as CD1a+ and MHC class II+ and CD19-. Cell densities in immunostained tissue sections were evaluated using a scale from 0 to +4. RESULTS: Immunostaining in control dystrophic corneas was negative for all antibodies. A moderate to high density of CD8+, CD14+, and CD68+ cells was observed in the majority of rejected non-inflamed as well as in rejected inflamed corneal buttons. Strong positivity for HLA-DP and HLA-DR molecules in the epithelium, stroma, and endothelium was also demonstrated. Weak positivity for CD4 and CD25 was observed in six of 15 and 11 of 15 rejected corneas, respectively. The presence of dendritic cells in the basal layer of the epithelium and in the stroma was observed in 50% of the grafts. CONCLUSIONS: A high frequency of macrophages, the presence of DC in the explants, and strong expression of HLA-DP and HLA-DR molecules on resident cells are characteristics of rejected corneal allografts, whether actively inflamed or not. The presence of DC in the stroma of the grafted cornea suggests that they may be mainly responsible for T cell activation and graft rejection since DC are known to be a 100-fold more potent than macrophages as APC.

Marrow-derived activated macrophages are required during the effector phase of experimental autoimmune uveoretinitis in rats.

PURPOSE: Experimental autoimmune uveoretinitis (EAU), an established model for human endogenous (autoimmune) posterior uveitis, is a CD4+ T cell-mediated disease inducible in Lewis rats by intradermal inoculation with retinal antigens. Immunohistochemical studies have previously documented the lymphocyte profiles during various stages of the disease process. The purpose of the present study was to investigate the role of macrophages in EAU. METHODS: EAU was induced in Lewis rats, and the effect of macrophage depletion, using the drug dichlorodimethylene diphosphonate (Cl2MDP) encapsulated in liposomes and administered intravenously, was assessed based on the clinical and histological profile of the disease. RESULTS: The results have shown that in control animals macrophages occur early, feature prominently throughout the course of the disease and display considerable heterogeneity: marrow-derived ED1+ cells and ED3+ cells are the major infiltrating cells, with many cells also expressing ED7 and ED8. In contrast, few cells expressed the ED2 antigen during EAU, even though ED2+ "resident" macrophages occur in the normal choroid. Macrophage depletion, using intravenously injected dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, caused a delay in the onset and a reduction in the severity of EAU when administered during the "effector" stage of the disease, i.e. 9-11 days after inoculation with retinal antigen. The delay in disease onset was greater when liposomes were mannosylated and was accompanied by a reduction in the overall inflammatory cell infiltrate into the eye and reduced tissue damage. In addition, there was a reduction in the level of expression of MHC Class II antigen and CR3 (ED7) antigen, a marker of macrophage activation, in Cl2MDP-treated animals compared to controls. CONCLUSION: These results suggest that blood-borne, activated macrophages are major effectors of tissue damage during EAU.

Localization and characterization of major histocompatibility complex class II-positive cells in the posterior segment of the eye: implications for induction of autoimmune uveoretinitis.

PURPOSE: To identify potential antigen-presenting cells in the choroid and retina of the normal rat eye, with a view to proposing a role for such cells in the induction and perpetuation of experimental autoimmune uveoretinitis, a model of human uveoretinal inflammation. METHODS: Immunohistochemical and electron microscopic studies using a panel of monoclonal antibodies were performed on frozen sections of the perfused-fixed normal Lewis rat eye, choroid whole mounts, and cytospin preparations of cells harvested from choroid/ciliary body explant cultures. In addition, time-lapse video recordings of migratory uveal tract cells in culture were taken. RESULTS: No major histocompatibility complex class II-positive cells were found in the normal Lewis rat retina. However, at least three populations of potential antigen-presenting cells were found in the uveal tissues of the eye: classical dendritic cells expressing high levels of major histocompatibility complex class II antigen; resident dendritiform macrophages, which were negative for major histocompatibility complex class II antigen, but expressed specific macrophage markers (ED2); and blood-borne macrophages (ED1) that had emigrated from the vasculature into the tissue compartment. In addition there were small numbers of cells expressing novel markers such as markers usually found only on macrophage subsets in splenic tissue (ED3) and a recently described marker for veiled dendritic cells (OX62). Dendritic cells and resident dendritiform macrophages closely interacted with each other and with tissue cells, particularly retinal pigment epithelial cells. CONCLUSIONS: The posterior uveal tract is richly populated with classical dendritic cells expressing constitutive high levels of major histocompatibility complex class II antigen. There are also several types of macrophages with the potential to modulate immune responses in the posterior segment. Interactions among these cells and with resident tissue cells such as retinal pigment epithelial cells are probably central to the initiation of (auto)immune responses in the posterior segment of the eye.

Adhesion molecule expression in acute and fibrotic sympathetic ophthalmia.

Samples of iris ciliary body, choroid and retina from normal eyes and from 2 cases of sympathetic ophthalmitis (one acute and one late stage fibrosis) were examined for the expression of the VLA integrins beta 1 and alpha 1-6, and the integrin beta 3, in addition to ICAM-1, VCAM-1, ELAM-1 and CD44 using an APAAP staining technique. The expression of VLA-4, VLA-5, VCAM-1, ICAM-1, and CD44 was significantly increased and ELAM-1 was slightly increased in acute sympathetic ophthalmitis in comparison to fibrotic and normal eyes. VLA-6 was moderately increased in acute and fibrotic cases and VLA-2 VLA-3 and beta 3 were moderately expressed on all tissues examined. The differential expression of molecules known to be involved in lymphocyte activation and adhesion in acute sympathetic ophthalmitis suggests that certain adhesion molecules play a role in the pathogenesis of intraocular inflammation and may be suitable targets for immunotherapy.

Predicting the occurrence of antibiotic-induced candidal vaginitis (AICV).

This paper reports the sensitivity, specificity, and predictive values of symptoms in the diagnosis of antibiotic-induced candidal vaginitis (AICV) among 74 women recruited from three primary care practices. All subjects, who were examined both pre- and post-antibiotic treatment for acute respiratory, urinary tract, or skin infections, were initially free of vaginitis. Twenty-four subjects developed candidal vaginitis (CV), indicated by vaginitis symptoms or signs and a positive candidal culture or KOH preparation; there were no mixed infections. Fifty women did not develop AICV and, of this group, four developed a nonyeast vaginitis. Aggregate symptoms (pruritus and/or discharge) had 87.5% sensitivity, 95.8% specificity, and positive and negative predictive values of 91.3% and 93.9%, respectively. These values are much higher than those reported in studies of CV that excluded women on antibiotics. We conclude that women who develop vaginitis symptoms while on short courses of antibiotics may be treated as AICV without confirmatory examination.

Monoclonal antibodies to cultured human glomerular mesangial cells. I. Reactivity with haematopoietic cells and normal kidney sections.

The aim of this study was to produce monoclonal antibodies to cultured human glomerular mesangial cells in order to obtain specific markers for these cells and to aid the study of their function. Using standard monoclonal antibody techniques, 29 hybridomas producing antibodies directed to cultured mesangial cells were obtained. Most of these antibodies were not reactive with normal or neoplastic haematopoietic cell lines by flow cytometry. Fourteen of the 29 culture supernatants bound to various components of normal human kidney sections stained by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. Ten of these supernatants reacted with components within the glomerulus, with six binding to the mesangium. These studies suggest that (1) mesangial cells in culture may show significant de-differentiation, because most supernatants which reacted with mesangial cells in culture did not do so in tissue sections; (2) antibodies reactive with haematopoietic cells may not detect the majority of immunogenic surface antigens on cells in tissues; and (3) some of the antibodies which we have produced may prove to be useful markers for mesangial cells in glomerular disease.

An analysis of T lymphocyte subpopulations in patients with Alzheimer's disease.

The relative proportions of regulatory T lymphocyte subpopulations and T lymphocyte proliferative responses to the mitogen phytohemagglutinin (PHA) were studied in 30 patients with clinically apparent Alzheimer's disease (mean age = 71.5). Comparisons were made with 30 age-matched, nondemented healthy controls (mean age = 68.8) and with 20 younger, normal adult controls (mean age = 27.5). The relative percentages of total peripheral T lymphocytes, T helper/inducer lymphocytes (Th), and T suppressor/cytotoxic lymphocytes (Ts) were similar between the Alzheimer patient group and the age-matched controls. However, when the two older groups were compared with the young adult controls, a significant decrease was seen in the percentage of Ts cells, with a concomitant increase in the ratio of Th:Ts. Lymphocyte proliferative responses to PHA were similar in the Alzheimer patients and their age-matched controls; however, when compared with the young adult controls, a significant decrease in responsiveness for both older groups was observed. These results confirm decreases in certain immune indices with aging, but suggest that there are no changes in T lymphocyte subsets or in lymphocyte proliferation, which are unique to Alzheimer's disease.

Effects of an educational intervention on the rate of recurrent urinary tract infections in selected female outpatients.

This study was designed to prospectively study the effect of health education on the recurrence rate of urinary tract infections in female outpatients. Thirty-four volunteers were randomly assigned to either an experimental education group or a control group. Controls were offered routine patient information provided by practitioners at the outpatient clinic. Members of the experimental group participated in an educational session which addressed urinary tract infections, its risk factors, and behavioral changes which might reduce its recurrence. At follow-up three months after the educational session, the experimental group had a statistically significant (p less than .05) reduction in the recurrence of urinary tract infections.