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Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.

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The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

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Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

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The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.

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Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation. Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal. In vivo, these complexes cluster predominantly in large groups at the cell poles. The function of chemoreceptor clustering is currently unknown. To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several Escherichia coli mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP. We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells. Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering. Nonchemotactic cells expressing only CheA(S), a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering. Cells expressing only full-length CheA (CheA(L)) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA(L) and CheA(S), demonstrating that CheA(L) alone can mediate polar clustering.

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Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway. These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE). In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus. Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells. Although these results do not indicate on obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.

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Several mathematical models to predict tissue adaptation have been derived since Julius Wolff proposed a function-form relationship for bone. These can be formulated as computational procedures (algorithms) to predict bone adaptation around implants. The objective of this paper was to further develop the damage-adaptive algorithm, to test its validity, and to determine the relationship between it and algorithms based on strain energy. This was achieved using finite element models of the proximal femur, one for the intact case and another for the case where a noncemented hip prosthesis is implanted. The finite models were generated using CT scan data. Initial bone resorption patterns around a femoral prosthesis following total hip arthroplasty were computed for both damage-adaptive and strain-adaptive adaptation rules. It is found that the damage-adaptive algorithm can successfully predict the bone's adaptive behaviour in response to altered mechanical loading provided that account is taken of the nonlinear nature of damage accumulation. Predictions are made using a strain energy stimulus for comparison with the damage stimulus, and a theoretical relationship between the two is proposed. It is shown that an advantage of the damage approach over the strain-based approach is that the nonlinearity required to replicate clinically observed resorption patterns can be derived theoretically, whereas for strain-adaptive remodelling, empirical relationships are assumed.

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The effect of bone-prosthesis bonding on proximal load transfer is investigated using a coupled experimental and finite element analysis on a synthetic femur. Three-dimensional finite element models for an intact femur and a femur implanted with a cementless prosthesis were constructed from the experimental models used, and the proximal femoral strains recorded for two loading conditions approximating a one-legged stance. The approach was used to investigate a press-fitted and a fully bonded bone-prosthesis structure to identify the stem-bone behaviour for both interface conditions and their implications for proximal bone load transfer. Regression slopes close to unity indicated that the finite element predictions were an accurate estimate of the experimental measurements. Physiological surface strains were recorded only when the abductor force was included in the loading. Meanwhile, experimental measurements and numerical predictions showed that a different load transfer pattern is to be expected for normally press-fitted and glued press-fitted stems. The finite element model for the treated femur, modelling both interface conditions correlated very well with the experimental model. These finite element models subsequently modified and used to analyse the effect of different interface conditions predicted a significant increase in the load transfer to the proximal calcar bone when only proximal bonding is achieved. This study suggests that information obtained for the assessment and prediction of total hip arthroplasty longevity by numerical and experimental techniques used together and in parallel is of greater value than either technique used alone. The employment of a femur analogue as featured in this study is also shown to be a suitable alternative to cadaveric specimens in such an analysis.

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CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.

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Evaluating the state of stress/strain for a given geometry and load in femurs can be done both experimentally, measuring strain at a limited number of locations, and theoretically with finite element models. Another approach is to describe the state of strain with a few synthetic indices. For this purpose the reverse elastic problem (i.e. bone parameters are estimated given the strain distribution and loads) needs to be solved as opposed to the finite element direct problem. Such reverse models can be then used: (1) to describe simply the strain distribution by means of few synthetic indices; (2) to explain the state of strain; and (3) to predict the strain distribution under different loading conditions. Various linear models, characterized by two to five bone related parameters, were tested on (1) 12 femurs, (2) a finite element model, and (3) data taken from the literature, for a total of 43 loading cases. Three and four-parameter models were able to fit the experimental strain distributions with mean squared residuals smaller than 5% of the strain range. The consistency of the model was proved by the repeatability of the parameters estimate for identical femurs. Furthermore, the bone-related coefficients were able to detect the stiffening effect of the implantation of an uncemented stem. Finally, the model can be used for predictive purposes if the parameter estimates are used with different loading conditions.

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Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in chemotactic signal transduction. As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB. The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation. However, it does retain a functional kinase domain. As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants. Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors. The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine. Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.

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The aim of this study was to determine the validity with which the finite element method could model synthetic bone and thereby determine the appropriateness of such femur analogues for application in pre-clinical tests. The performance of these synthetic femora was compared with cadaveric bone when employing the same geometric and material definition protocols. A four-point bend loading configuration was selected for this analysis. Four synthetic femurs and an embalmed cadaveric bone were tested experimentally to determine the structural bending stiffness (k) for the diaphysis of these bones. A finite element (FE) model was generated and an analysis performed for each bone type to estimate the Young's modulus (E) required to obtain a model stiffness equivalent to that obtained experimentally. The estimated material elastic modulus in the FE model for the synthetic femur was found to be very similar to available data for this bone analogue. The estimated cadaveric bone modulus however was found to differ significantly from documented values for cortical bone. A theoretical analysis demonstrated the great sensitivity of the estimated modulus value to the accuracy of the geometric definition. The very low variability found in the experimental test on the synthetic bones together with their more regular geometry and the possibility of achieving greater accuracy in geometric definition was shown to enable the production of a valid FE model of this bone for an isotropic homogeneous material description. Conversely, the greater irregularity of geometry, together with the less obvious differentiation between the cortical and cancellous bone in the cadaveric specimen makes accurate geometric description of this bone very difficult. This fact, together with the uncertainty concerning the quality of the cadaveric bone and its viscoelastic response during mechanical testing, makes reproduction of its behaviour in a FE model a much more demanding task. It is suggested that this greater capability of reproducing the experimental behaviour of the synthetic bone makes them a very useful model for both experimental and numerical studies which involve in-vitro pre-clinical testing of implant design and stem-bone behaviour.

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A method for the prediction of the time-course of bone adaptation based on an alternative hypothesis of strength optimization has been previously investigated and developed by Prendergast and Taylor. This paper extends our work in the study of the effectiveness of this bone adaptation model in predicting similar bone remodelling to that observed in animal experiments. In particular the experimental work which has been modelled is that of Lanyon, Goodship, Pye and McFie. An anatomical finite element model of the sheep's forelimb has been generated for this purpose and is used to estimate stresses in the bone structure for the normal and osteotomized condition. The propensity for remodelling of the altered bone structure is predicted using the proposed remodelling law for the new stress field in the bone structure. The preliminary results indicate an initial bone adaptation pattern similar to that observed experimentally without the necessity to use arbitrarily different constants for the endosteal and periosteal surfaces. We therefore suggest that the remodelling law based on damage and repair gives a better predictive model of bone adaptation than previous models.

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Vinyl chloride monomer (VCM), already identified as a human animal carcinogen, was selected as a model agent to explore an area of concern for single and intermittent low level exposure. In traditional cancer bioassay, animals are repeatedly exposed over their lifespan to a dose of suspected chemical. In the current studies rats and mice were exposed in an inhalation chamber to single one-hour doses of VCM ranging from 50 to 50,000 ppm. A second group was given 10 one-hour exposures to 500 ppm or 100 one-hour exposures to 50 ppm of the same chemical. All animals were then observed for the remainder of their lives, generally 18-24 months. Moribund animals were euthanized, and survivors were sacrificed on schedule and their tissues examined for pathological changes. Specifically, the oncogenic study demonstrated dose related effects for single one-hour exposure of VCM at high levels, i.e., 5,000 and 50,000 ppm. These concentrations increased the incidence of pulmonary adenomas and carcinomas in mice. Repeated exposure of A/J mice to the same chemical at 500 ppm X 10 one-hour exposures also increased the incidence of pulmonary adenomas and carcinomas which are considered highly one-hour exposure, no significant increase in tumors was observed. Rats exposed to identical concentrations of VCM failed to elicit a tumorigenic response.

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