Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Intravenous mesenchymal stem cell therapy for traumatic brain injury.

OBJECT: Cell therapy has shown preclinical promise in the treatment of many diseases, and its application is being translated to the clinical arena. Intravenous mesenchymal stem cell (MSC) therapy has been shown to improve functional recovery after traumatic brain injury (TBI). Herein, the authors report on their attempts to reproduce such observations, including detailed characterizations of the MSC population, non-bromodeoxyuridine-based cell labeling, macroscopic and microscopic cell tracking, quantification of cells traversing the pulmonary microvasculature, and well-validated measurement of motor and cognitive function recovery. METHODS: Rat MSCs were isolated, expanded in vitro, immunophenotyped, and labeled. Four million MSCs were intravenously infused into Sprague-Dawley rats 24 hours after receiving a moderate, unilateral controlled cortical impact TBI. Infrared macroscopic cell tracking was used to identify cell distribution. Immunohistochemical analysis of brain and lung tissues 48 hours and 2 weeks postinfusion revealed transplanted cells in these locations, and these cells were quantified. Intraarterial blood sampling and flow cytometry were used to quantify the number of transplanted cells reaching the arterial circulation. Motor and cognitive behavioral testing was performed to evaluate functional recovery. RESULTS: At 48 hours post-MSC infusion, the majority of cells were localized to the lungs. Between 1.5 and 3.7% of the infused cells were estimated to traverse the lungs and reach the arterial circulation, 0.295% reached the carotid artery, and a very small percentage reached the cerebral parenchyma (0.0005%) and remained there. Almost no cells were identified in the brain tissue at 2 weeks postinfusion. No motor or cognitive functional improvements in recovery were identified. CONCLUSIONS: The intravenous infusion of MSCs appeared neither to result in significant acute or prolonged cerebral engraftment of cells nor to modify the recovery of motor or cognitive function. Less than 4% of the infused cells were likely to traverse the pulmonary microvasculature and reach the arterial circulation, a phenomenon termed the "pulmonary first-pass effect," which may limit the efficacy of this therapeutic approach. The data in this study contradict the findings of previous reports and highlight the potential shortcomings of acute, single-dose, intravenous MSC therapy for TBI.

Characterization of the inflammatory cells in ascending thoracic aortic aneurysms in patients with Marfan syndrome, familial thoracic aortic aneurysms, and sporadic aneurysms.

OBJECTIVE: This study sought to characterize the inflammatory infiltrate in ascending thoracic aortic aneurysm in patients with Marfan syndrome, familial thoracic aortic aneurysm, or nonfamilial thoracic aortic aneurysm. BACKGROUND: Thoracic aortic aneurysms are associated with a pathologic lesion termed "medial degeneration," which is described as a noninflammatory lesion. Thoracic aortic aneurysms are a complication of Marfan syndrome and can be inherited in an autosomal dominant manner of familial thoracic aortic aneurysm. METHODS: Full aortic segments were collected from patients undergoing elective repair with Marfan syndrome (n = 5), familial thoracic aortic aneurysm (n = 6), and thoracic aortic aneurysms (n = 9), along with control aortas (n = 5). Immunohistochemistry staining was performed using antibodies directed against markers of lymphocytes and macrophages. Real-time polymerase chain reaction analysis was performed to quantify the expression level of the T-cell receptor beta-chain variable region gene. RESULTS: Immunohistochemistry of thoracic aortic aneurysm aortas demonstrated that the media and adventitia from Marfan syndrome, familial thoracic aortic aneurysm, and sporadic cases had increased numbers of T lymphocytes and macrophages when compared with control aortas. The number of T cells and macrophages in the aortic media of the aneurysm correlated inversely with the patient's age at the time of prophylactic surgical repair of the aorta. T-cell receptor profiling indicated a similar clonal nature of the T cells in the aortic wall in a majority of aneurysms, whether the patient had Marfan syndrome, familial thoracic aortic aneurysm, or sporadic disease. CONCLUSION: These results indicate that the infiltration of inflammatory cells contributes to the pathogenesis of thoracic aortic aneurysms. Superantigen-driven stimulation of T lymphocytes in the aortic tissues of patients with thoracic aortic aneurysms may contribute to the initial immune response.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Plant regeneration from an endangered valuable cork oak tree by somatic embryogenesis.

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

Diseño y evaluación de un procedimiento on-line para la biorremediación de aguas radiactivas en centrales nucleares

Recientemente se ha demostrado la existencia de microorganismos en las piscinas de almacenamiento de combustible nuclear gastado en las centrales nucleares utilizando técnicas convencionales de cultivo en el laboratorio. Estudios posteriores han puesto de manifiesto que los microorganismos presentes eran capaces de colonizar las paredes de acero inoxidable de las piscinas formando biopelículas. Adicionalmente se ha observado la capacidad de estas biopelículas de retener radionúclidos, lo que hace pensar en la posibilidad de utilizarlas en la descontaminación de las aguas radiactivas de las piscinas. En la presente tesis se plantea conocer más profundamente la biodiversidad microbiana de las biopelículas utilizando técnicas de biología molecular como la clonación, además de desarrollar un sistema de descontaminación a escala piloto con el objetivo de valorar si el proceso podría resultar escalable a nivel industrial. Para ello se diseñaron y fabricaron dos biorreactores en acero inoxidable compatibles con las condiciones específicas de seguridad sísmica y protección frente a la radiación en la zona controlada de una central nuclear. Los biorreactores se instalaron en la Central Nuclear de Cofrentes (Valencia) en las proximidades de las piscinas de almacenamiento de combustible nuclear gastado y precediendo a las resinas de intercambio iónico, de forma que reciben el agua de las piscinas permitiendo el análisis in situ de la radiación eliminada del agua de las mismas. Se conectó una lámpara de luz ultravioleta a uno de los biorreactores para poder comparar el desarrollo de bipelículas y la retención de radiactividad en ambas condiciones. En estos biorreactores se introdujeron ovillos de acero inoxidable y de titanio que se extrajeron a diversos tiempos, hasta 635 días para los ovillos de acero inoxidable y hasta 309 días para los ovillos de titanio. Se analizaron las biopelículas desarrolladas sobre los ovillos por microscopía electrónica de barrido y por microscopía de epifluorescencia. Se extrajo el ADN de las biopelículas y, tras su clonación, se identificaron los microorganismos por técnicas independientes de cultivo. Asimismo se determinó por espectrometría gamma la capacidad de las biopelículas para retener radionúclidos. Los microorganismos radiorresistentes identificados pertenecen a los grupos filogenéticos Alpha-proteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus y Bacteroidetes. Las secuencias de estos microorganismos se han depositado en el GenBank con los números de acceso KR817260-KR817405. Se ha observado una distribución porcentual ligeramente diferente en relación con el tipo de biorreactor. Las biopelículas han retenido fundamentalmente radionúclidos de activación. La suma de Co-60 y Mn-54 ha llegado en ocasiones al 97%. Otros radionúclidos retenidos han sido Cr-51, Co-58, Fe-59, Zn-65 y Zr-95. Se sugiere un mecanismo del proceso de retención de radionúclidos relacionado con el tiempo de formación y desaparición de las biopelículas. Se ha valorado que el proceso escalable puede ser económicamente rentable. ABSTRACT The existence of microorganisms in spent nuclear fuel pools has been demonstrated recently in nuclear power plants by using conventional microbial techniques. Subsequent studies have revealed that those microorganisms were able to colonize the stainless steel pool walls forming biofilms. Additionally, it has been observed the ability of these biofilms to retain radionuclides, which suggests the possibility of using them for radioactive water decontamination purposes. This thesis presents deeper knowledge of microbial biofilms biodiversity by using molecular biology techniques such as cloning, and develops a decontamination system on a pilot scale, in order to assess whether the process could be scalable to an industrial level. Aiming to demonstrate this was feasible, two stainless steel bioreactors were designed and manufactured, both were compatible with seismic and radiation protection standards in the controlled zone of a nuclear plant. These bioreactors were installed in the Cofrentes Nuclear Power Plant (Valencia) next to the spent nuclear fuel pools and preceding (upstream) ion exchange resins. This configuration allowed the bioreactors to receive water directly from the pools allowing in situ analysis of radiation removal. One ultraviolet lamp was connected to one of the bioreactors to compare biofilms development and radioactivity retention in both conditions. Stainless steel and titanium balls were introduced into these bioreactors and were removed after different time periods, up to 635 days for stainless steel balls and up to 309 days for titanium. Biofilms developed on the balls were analyzed by scanning electron microscopy and epifluorescence microscopy. DNA was extracted from the biofilms, was cloned and then the microorganisms were identified by independent culture techniques. Biofilms ability to retain radionuclides was also determined by gamma spectrometry. The identified radioresistant organisms belong to the phylogenetic groups Alphaproteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus and Bacteroidetes. The sequences of these microorganisms have been deposited in GenBank (access numbers KR817260-KR817405). A different distribution of microorganisms was observed in relation to the type of bioreactor. Biofilms have essentially retained activation radionuclides. Sometimes the sum of Co-60 and Mn-54 reached 97%. Cr-51, Co-58, Fe-59, Zn-65 and Zr-95 have also been retained. A radionuclide retention process mechanism related to biofilms formation and disappearance time is suggested. It has been assessed that the scalable process can be economically profitable.

Breakdown of plant cell biomass by filamentous fungi

Three species of filamentous fungi, Botrytis cinerea, Sporotrichum thermophile and Trichoderma viride, have been selected to assess the potential of utilizing filamentous fungi to degrade plant cell biomass produced by mass cell culture techniques. All three fungal species grew comparatively well on plant cell biomass with no requirement for supplementary nutrients. Of the three species assessed B. cinerea demonstrated the most growth. This species also produced the greatest yield of D-glucose. However, when culture conditions were modified, yields of D-glucose were markedly reduced indicating that the combination of species and culture conditions must be thoroughly investigated to ensure maximum product yield. The growth of filamentous fungi on plant cells also markedly affected the nature of the resulting fungal-plant cell residue, increasing the levels of soluble carbohydrates and essential amino acids with the largest increase in these materials being promoted by B. cinerea.

Cells, surfaces and adhesion

This thesis concerns cell adhesion to polymer surfaces with an experimental emphasis on hydrogels. The thesis begins with a review of the literature and a synthesis of recent evidence to describe the process of cell adhesion in a given situation. The importance of understanding integrin-adhesion protein interactions and adhesion protein-surface interactions is emphasised. The experimental chapters describe three areas of investigation. Firstly, in vitro cell culture techniques are used to explore a variety of surfaces including polyethylene glycol methacrylate (PEGMA) substituted hydrogels, sequence distribution modified hydrogels and worn contact lenses. Cell adhesion to PEGMA substituted gels is found to decrease with increases in polyethylene oxide chain length and correlations are made between sequence distribution and adhesion. Worn contact lenses are investigated for their cell adhesion properties in the presence of antibodies to specific adhesion proteins, demonstrating the presence of vitronectin and fibronectin on the lenses. The second experimental chapter addresses divalent cation regulation of integrin mediated cell adhesion. Several cell types and various cations are used. Zinc, previously not regarded as an important cation in the process, is found to inhibit 3T3 cell adhesion to vitronectin that is promoted by other divalent cations. The final experimental chapter concerns cell adhesion and growth on macroporous hydrogels. A variety of freeze-thaw formed porous gels are investiated and found generally to promote cell growth rate.Interpenetrating networkbased gels (IPN) are made porous by elution of dextrin particles of varying size and loading density. These materials provide the basis for synthetic cartilage. Cartilage cells (chondrocytes) plated onto the surface of the porous IPN materials maintain a rounded shape and hence phenotypic function when a critical pore size and density is achieved. In this way, a prospective implant, made porous at the perpendicular edges contacting natural cartilage can be both mechanically stabilised and encourage the maintenance of normal matrix production at the tissue interface.

Towards an improved ocular drug delivery system

The ultimate aim of this project was to design new biomaterials which will improve the efficiency of ocular drug delivery systems. Initially, it was necessary to review the information available on the nature of the tear fluid and its relationship with the eye. An extensive survey of the relevant literature was made. There is a common belief in the literature that the ocular glycoprotein, mucin, plays an important role in tear film stability, and furthermore, that it exists as an adherent layer covering the corneal surface. If this belief is true, the muco-corneal interaction provides the ideal basis for the development of sustained release drug delivery. Preliminary investigations were made to assess the ability of mucin to adhere to polymer surfaces. The intention was to develop a synthetic model which would mimic the supposed corneal/mucin interaction. Analytical procedures included the use of microscopy (phase contrast and fluorescence), fluorophotometry, and mucin-staining dyes. Additionally, the physical properties of tears and tear models were assessed under conditions mimicking those of the preocular environment, using rheological and tensiometric techniques. The wetting abilities of these tear models and opthalmic formulations were also investigated. Tissue culture techniques were employed to enable the surface properties of the corneal surface to be studied by means of cultured corneal cells. The results of these investigations enabled the calculation of interfacial and surface characteristics of tears, tear models, and the corneal surface. Over all, this work cast doubt on the accepted relationship of mucin with the cornea. A corneal surface model was designed, on the basis of the information obtained during this project, which would possess similar surface chemical properties (i.e. would be biomimetic) to the more complex original. This model, together with the information gained on the properties of tears and solutions intended for ocular instillation, could be valuable in the design of drug formulations with enhanced ocular retention times. Furthermore, the model itself may form the basis for the design of an effective drug-carrier.

Studies on the composition and nutritional value of the cultivated mushroom, agaricus bisporus (Lange) Sing

The effects of various cultural conditions on the composition and nutritional quality of Agaricus bisporus (Lange) Sing. were investigated. Variation in composition was found between different classes of sample. Sampling techniques were standardised to allow for major variations in the different developmental stages and culture ages. Fruitbodies were found to be of low calorific value but contained protein of high digestibility and quality, containing all the essential amino acids required by man. Quantitative estimates of the sulphur-containing amino acids indicated that fruitbodies were deficient in methionine and cysteine. The extent of water application and the supplementation of conventional substrates with various nitrogen-containing substances, influenced yield and composition, establishing the importance of these two factors in the physiology of fruitbodies and cultural management. Storage conditions influenced composition, high temperatures being deleterious to the nutritional value of fruitbodies. Submerged culture techniques were used to investigate the effects of various nutrients on growth and composition of mushroom mycelium, with special reference to the sulphur-containing amino acids. Yield and composition were greatly affected by the carbon:nitrogen ratio of the medium and by the nitrogen source. Significant increases in mycelial methionine content were observed on the addition of inorganic sulphate, the methionine derivative N-acetyl-L-methionine, and L-methionine. A greater increase in methionine content was obtained when the biomass of a thermophilic bacterium isolated from compost was used as a nitrogen source.

A potentially scalable method for the harvesting of hMSCs from microcarriers

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too. © 2014 The Authors.

Human brain slices for epilepsy research:pitfalls, solutions and future challenges

Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Cortactin recruitment by enterohemorrhagic Escherichia coli O157:H7 during infection in vitro and ex vivo

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF(U). Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.