Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Functional characterization, homology modeling and docking studies of beta-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

Glycosyl hydrolase family 1 beta-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving beta-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this beta-glucosidase were found to be 45 A degrees C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K (m) and V (max) were found to be 38.59 mu M and 0.8237 mu M/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 mu M and 0.1037 mu M/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (Delta G) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

Structural insights into N-terminal to C-terminal interactions and implications for thermostability of a (beta/alpha)(8)-triosephosphate isomerase barrel enzyme

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)(8)-triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 degrees C to 75 degrees C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 degrees C to 68 degrees C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. Database The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis

Background: The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear. Methods: The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 mu m in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence. Results: Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. Conclusion: 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Influence of Extracellular Matrix Components on the Expression of Integrins and Regeneration of Adult Retinal Ganglion Cells

Purpose Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites? Methods Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against beta III-Tubulin to identify the RGCs, and antibodies against the integrin subunits: alpha V, alpha 1, alpha 3, alpha 5, beta 1 or beta 3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed. Results PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly alpha 1 beta 1 or alpha 3 beta 1 on L, alpha 1 beta 1 on CI and CIV, and alpha 5 beta 3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK). Conclusions Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

Efeito do Sistema de Secreção do Tipo VI de Pseudomonas aeruginosa em modelo murino de pneumonia aguda

O Sistema de Secreção do Tipo VI (SST6), o mais recente maquinário de secreção descrito em bactérias Gram-negativas, é amplamente distribuído entre as diversas espécies deste grupo de microrganismos. Esse aparato de secreção é capaz de injetar efetores proteicos em células alvo, eucarióticas e procarióticas. Estudos sobre o papel do SST6 na virulência microbiana revelaram que este sistema secretório participa ativamente do estabelecimento de infecções, contribuindo para a sobrevivência das bactérias no interior de fagócitos. O genoma da cepa PAO1 de Pseudomonas aeruginosa apresenta três loci que codificam aparatos de SST6, denominados de H1-SST6, H2-SST6 e H3-SST6, Porém, pouco se sabe sobre a participação do SST6 na patogênese de infecções por P. aeruginosa. Assim, o presente estudo investigou o papel de H1-SST6, H2-SST6 e H3-SST6 durante a infecção pulmonar aguda de camundongos. Para isso, camundongos C57/BL6 foram infectados com diferentes doses de bactérias da cepa selvagem PAO1 ou das cepas mutantes PAO1∆H1, PAO1∆H2, PAO1∆H3 ou PAO1∆H1∆H2∆H3. Após 24 horas, os lavados broncoalveolares (LBAs) de animais controle e infectados foram recuperados para a contagem de leucócitos totais e polimorfonucleares e para a quantificação, por ELISA, da quimiocina para neutrófilos, KC, e das citocinas pró-inflamatórias IL-1β e TNF-α. Em outros experimentos, os pulmões, fígados, baços e rins dos animais foram macerados para a pesquisa da carga bacteriana e da disseminação sistêmica das bactérias. A citotoxicidade do SST6 foi determinada, in vitro, em neutrófilos humanos, pela marcação com iodeto de propídeo (PI) e anexina-V seguida da análise em citometria de fluxo. Os resultados mostraram que a inativação dos três SST6 reduziu significativamente a concentração de neutrófilos nos LBAs quando os animais foram infectados com 10⁷ Unidades Formadoras de Colônias de P. aeruginosa. Nesta dose, foi observado que as medianas do número de bactérias detectadas nos animais infectados com as mutantes no SST6 foram menores do que as detectadas nos animais infectados com a cepa parental PAO1. As mutações no SST6 não afetaram a disseminação sistêmica da bactéria. A pesquisa da secreção de citocinas pró-inflamatórias mostrou que, embora tenha sido observada uma redução nas medianas das concentrações de TNF-α nos LBAs de camundongos infectados com a cepa PAO1∆H1∆H2∆H3, em relação aos LBAs de camundongos infectados com a cepa parental, essa diferença não foi significativa. Como a pesquisa de IL-1β e KC não contribuiu para a elucidação dos mecanismos envolvidos na redução da concentração de neutrófilos nos LBAs dos camundongos infectados pela cepa tripla mutante, foi pesquisado o possível efeito do SST6 na morte de neutrófilos humanos. Os resultados mostraram que não houve diferenças significativas quando as diferentes amostras de células infectadas foram comparedas entre si. Em conclusão, os resultados do presente estudo mostraram que o SST6 pode interferir na resposta de neutrófilos durante a pneumonia aguda, mas estudos adicionais são necessários para determinar o papel deste mecanismo de secreção na patogênese de P. aeruginosa.

Rosuvastatina, doença hepática gordurosa não alcoólica e ativação das células estreladas hepáticas em camundongos

Introdução: A atual epidemia de obesidade tem chamado a atenção para a doença hepática gordurosa não alcoólica (DHGNA). Atualmente não existe um medicamento para o tratamento da esteatose hepática, embora as estatinas sejam muito prescritas para pacientes obesos, este medicamento destina-se ao tratamento da hipercolesterolemia. Este trabalho teve como objetivo investigar os efeitos da rosuvastatina em um modelo de obesidade induzida por dieta, como foco principal a DHGNA e os marcadores hepáticos da lipogênese e beta-oxidação e ativação de células estreladas hepáticas (CEHs) em camundongos. Métodos: Camundongos machos C57BL/6 receberam dieta padrão (SC, 10% de energia como lipídios) ou dieta rica em gorduras (HF, 50% de energia como lipídios) durante 12 semanas. Em seguida, 7 semanas de tratamento, foram feitas, formando os grupos: SC, SCR (SC + rosuvastatina), HF e HFR (HF + rosuvastatina). As análises bioquímicas e técnicas moleculares foram aplicadas para abordar os resultados plasmáticos e moleculares. Resultados: O grupo HF apresentou maiores valores de insulina, colesterol total, triglicerídeos e leptina que o grupo SC, todos os quais foram reduzidos significativamente após o tratamento com Rosuvastatina no grupo HFR. O grupo HF apresentou maior percentual de esteatose, assim como maior ativação das CEHs, enquanto que a rosuvastatina provocou uma redução de 21% na esteatose hepática e atenuou a ativação das CEHs no grupo HFR. Em concordância com os achados histológicos, as expressões de SREBP-1 e PPAR-gama foram aumentados nos animais HF e reduzido após o tratamento no grupo HFR. Por outro lado, a expressão reduzida de PPAR-alfa e CPT-1 foram encontrados nos animais HF, sendo tais parâmetros restaurados após o tratamento no grupo HFR. Conclusão: A rosuvastatina atenua significativamente a ativação das células estreladas na obesidade induzida por dieta, afetando o equilíbrio dos PPARs na lipotoxicidade. Diante desses achados a Rosuvastatina pode ser indicada como alternativa para auxiliar o tratamento da esteatose hepática.

The search for the IFN-gamma receptor in fish: Functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFN gamma R1 and IFN gamma R2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFN gamma Rs in fish. They are widely expressed in tissues, with IFN gamma R1 typically more highly expressed than IFN gamma R2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1 beta down regulating IFN gamma R1 expression but up regulating IFN gamma R2 expression. Overexpression of the two receptor chains in RTG-2 cells revealed that the level of IFN gamma R2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gamma IP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFN gamma R may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction. (c) 2009 Elsevier Ltd. All rights reserved.

Impact of wide bandgap p-type nc-Si on the performance of a-Si solar cells

This paper reports the impact of a wide bandgap p-type hydrogenated nanocrystalline silicon (nc-Si:H) on the performances of hydrogenated amorphous silicon (a-Si:H) based solar cells. The player consists of nanometer-sized Si crystallites and has a wide effective bandgap determined mainly by the quantum size-confinement effect (QSE). By incorporation of this p-layer into the devices we have obtained high performances of a-Si:H top solar cells with V-infinity=1.045 V and FF=70.3 %, and much improved mid and bottom a-SiGe:H cells, deposited on stainless steel (SS) substrate. The effects of the band-edge mismatch at the p/i-interface on the I-V characteristics of the solar cells arc discussed on the bases of the density-functional approach and the AMPS model.

Impact of wide bandgap p-type nc-Si on the performance of a-Si solar cells

This paper reports the impact of a wide bandgap p-type hydrogenated nanocrystalline silicon (nc-Si:H) on the performances of hydrogenated amorphous silicon (a-Si:H) based solar cells. The player consists of nanometer-sized Si crystallites and has a wide effective bandgap determined mainly by the quantum size-confinement effect (QSE). By incorporation of this p-layer into the devices we have obtained high performances of a-Si:H top solar cells with V-infinity=1.045 V and FF=70.3 %, and much improved mid and bottom a-SiGe:H cells, deposited on stainless steel (SS) substrate. The effects of the band-edge mismatch at the p/i-interface on the I-V characteristics of the solar cells arc discussed on the bases of the density-functional approach and the AMPS model.

SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.