To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

We compared temporal changes in isometric tension in cultured human umbilical vein endothelial cells inoculated on a polymerized collagen membrane with changes in cell-cell and cell-matrix adhesion derived by a mathematical model of transendothelial cell resistance. Thrombin and histamine disrupt barrier function by targeting a greater loss in cell-cell adhesion, which preceded losses in overall transendothelial resistance. There were minor losses in cell-matrix adhesion, which was temporally slower than the decline in the overall transendothelial resistance. In contrast, thrombin and histamine restored barrier function by initiating a restoration of cell-matrix adhesion, which occurred before an increase in overall transendothelial resistance. Thrombin mediated a second and slower decline in cell-cell adhesion, which was not observed in histamine-treated cells. This decline in cell-cell adhesion temporally correlated with expressed maximal levels of tension development, suggesting that actin-myosin contraction directly strains cell-cell adhesion sites. Pretreatment of cells with ML-7 mediated more rapid recovery of cell-cell adhesion and had no effect on cell-matrix adhesion. Taken together, expression of actin-myosin contraction affects the restoration of barrier function by straining cell-cell adhesion sites.

We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.

Dear Sir,The paradigm shift that has occurred in the last decade attributing most, if not all, cervical cancers to infection with human papillomavirus (HPV) has renewed efforts to control cervical cancer, especially in developing countries where it remains the leading cause of cancer deaths among women. Primary and secondary prevention efforts have begun to focus on detection and control of the virus, specifically HPV DNA testing for screening [1][2][3][4][5][6][7] and HPV vaccine development for prevention. 8 Critical to the success of HPV-based prevention efforts is a comprehensive spectrum of targeted genotypes, given that at least 10 different HPV types have been classified as group 1 human carcinogens. 9 The International Biological Study of Invasive Cervical Cancer (IBSCC) demonstrated that certain HPV genotypes, namely, HPV-16, -18, -31 and -45, accounted for 80% of the sampled invasive cancers from 21 countries. 10,11 Based on these results, vaccine efforts are targeted first to HPV-16, with the hope of reducing the cervical cancer burden by up to 50%, presumably with vaccines targeting HPV-18, -31 and -45 to follow. However, a study in Mozambique found that HPV-35 was the most prevalent genotype, both in all HPV-positive women (16.7 %) and among women with cervical neoplasia (18.4%). 12 It is important to determine if this is a geographically isolated finding or if the relative prevalence of HPV types attributable to cervical cancer development differs in sub-Saharan Africa, where primary prevention offers the greatest promise of control. We report the genotype distribution of HPV from a nested case-control study of women originally enrolled in a visual inspection with acetic acid (VIA) screening study in Harare, Zimbabwe. 13 Study participants were drawn from subjects enrolled in phase II VIA screening study conducted jointly by the University of Zimbabwe in Harare and the JHPIEGO Corporation, a Johns Hopkins University affiliate based in Baltimore, MD, USA. Details of subject recruitment have been described elsewhere. 13 Briefly, subjects enrolled in phase II of the VIA screening study were recruited from October 1996 through August 1997 among women aged 25-55 years attending 15 primary-care clinics in Chitungwiza and the greater Harare area of Zimbabwe. All enrolled women provided verbal informed consent, and the institutional review boards of both participating institutions approved study protocols. Participants were interviewed using a standardized questionnaire to assess demographics. Following the interview, each participant consented to a pelvic exam with collection of cells for Pap smear and HPV DNA testing. VIA screening was performed last. All participants were offered a colposcopic examination of the cervix, and biopsies were collected if indicated, usually on the same day. At the colposcopy visit, consenting women (23%) provided an oral mucosal specimen (OraSure; Epitope, Beaverton, OR) for HIV antibody testing (duplicate testing via commercially available ELISA; Organon Teknica, Durham, NC...

Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.