The effect of inflammatory agents on the blood-retinal barrier

The blood-retinal barrier (BRB), like that of the blood-brain barrier (BBB), forms a selective interface between the blood and the neural parenchyma. During inflammatory diseases of the retina there is a large scale increase in leucocyte infiltration and breakdown of the BRB. It is not entirely clear, however, what the causative factors are in BRB disruption, but recently cytokines have been implicated. An understanding of the role of cytokines within retinal inflammatory diseases, such as posterior uveitis where BRB breakdown leads to macular oedema and impaired vision, may lead to improved therapies and disease control. Cytokines have been detected within the eyes of patients with uveitis. Also, the injection of cytokines to experimental animals has been shown to cause leucocyte recruitment and an increase m vascular permeability. However, it is not known whether cytokines have a direct effect on increasing vascular permeability, if they induce the release of vasoactive mediators, or if they exert their effects through leucocyte recruitment. It was therefore hypothesised that the injection of a proinflammatory cytokine to the vitreous of the Lewis rat will induce a leucocytic infiltration to the retina and breakdown of the BRB. Inhibitor studies may then identify the mechanism by which BRB dysfunction is occurring. The effect of the proinflammatory cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) upon the Lewis rat BRB were investigated. Following an intravitreal injection of cytokine the structural integrity of the retina was evaluated using both light and electron microscopy. The permeability of the BRB was assessed by either introducing the small molecular weight tracer [14C]-mannitol, or the large molecular weight tracer horseradish peroxidase, into the circulation. Following administration of IL-1β a biphasic opening of the BRB was found, as determined by increased [14C]-mannitol extravasation. The initial breakdown occurred at 4 hr post injection (PI), corresponding to the appearance of leucocytes within the retinal vessels and adhering to the endothelium. The BRB permeability then decreased although this did not correspond to a reduction in the number of infiltrating leucocytes. A second, larger, and more prolonged increase in barrier permeability was detected at 24 to 48 hr PI which coincided with peak infiltration of leucocytes. Inhibitor and leucocyte depletion studies indicated that the IL-1β-induced breakdown of the BRB was mediated by histamine and cyclooxygenase metabolites (prostaglandins and thromboxanes), and associated with leucocyte infiltration to the retina. Following the intravitreal injection of TNF-α a monophasic and reversible opening of the BRB occurred but without the accompanying large scale cellular infiltrate. Immunohistochemical studies revealed the upregulation of major histocompatibility complex (MHC) class II molecules within the retina of TNF-α injected eyes. It was also demonstrated that there was an opening of the BRB of the non-injected contralateral eye which may be due to a neuronal reflex arc mechanism. Administration of IL-6 caused a minimal inflammatory cell infiltrate, but did not increase the permeability of the BRB at the dose examined. The intravitreal injection of the cytokines examined in this study exhibited different effects on the retina and BRB of the Lewis rat. IL-1β caused a biphasic breakdown of the BRB that appeared to be the consequence of classic inflammatory mediators and dependent on leucocyte infiltration, whereas TNF-α caused a monophasic increase in BRB permeability which appeared to be independent of leucocytic involvement. IL-6 had no effect on barrier integrity. Therefore, it appears that, in vivo, proinflammatory cytokines differ in their ability to induce leucocyte recruitment and cause increased vascular permeability. The mechanism by which TNF-α caused BRB breakdown is unknown, but the IL-1β induced biphasic breakdown of the BRB is dependent on the presence of leucocytes recruited to the retina and the release of vasoactive mediators such as histamine

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4(th) pharyngeal arch artery morphogenesis

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in 22q11 deletion syndrome patients and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9 deficient mice are born with complex cardiovascular malformations affecting the outflow tract and aortic arch arteries with failure of the 3(rd) and 4(th) pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared to Tbx1 heterozygous mice. Using a novel Pax9Cre allele we demonstrated that the site of this Tbx1-Pax9 genetic interaction is in the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for critical tissue interactions during normal morphogenesis of the pharyngeal arch artery system

Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique

BACKGROUND: Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. RESULTS: We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development. CONCLUSIONS: Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases

Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency

AIMS: Deletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal-->Pitx2c pathway controlling left-right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency. METHODS AND RESULTS: A Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. beta-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2(+/-) compared with wild-type (P = 2.62 x 10(-6)). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2(+/-) (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2(-/-) hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2(+/-) embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2(+/-) hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected. CONCLUSION: These results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left-right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin

Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world’s population. The small intestine is the parasite’s primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitro invasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells

Comparative genome analysis and genome-guided physiological analysis of Roseobacter litoralis

<p>Abstract</p> <p>Background</p> <p><it>Roseobacter litoralis </it>OCh149, the type species of the genus, and <it>Roseobacter denitrificans </it>OCh114 were the first described organisms of the <it>Roseobacter </it>clade, an ecologically important group of marine bacteria. Both species were isolated from seaweed and are able to perform aerobic anoxygenic photosynthesis.</p> <p>Results</p> <p>The genome of <it>R. litoralis </it>OCh149 contains one circular chromosome of 4,505,211 bp and three plasmids of 93,578 bp (pRLO149_94), 83,129 bp (pRLO149_83) and 63,532 bp (pRLO149_63). Of the 4537 genes predicted for <it>R. litoralis</it>, 1122 (24.7%) are not present in the genome of <it>R. denitrificans</it>. Many of the unique genes of <it>R. litoralis </it>are located in genomic islands and on plasmids. On pRLO149_83 several potential heavy metal resistance genes are encoded which are not present in the genome of <it>R. denitrificans</it>. The comparison of the heavy metal tolerance of the two organisms showed an increased zinc tolerance of <it>R. litoralis</it>. In contrast to <it>R. denitrificans</it>, the photosynthesis genes of <it>R. litoralis </it>are plasmid encoded. The activity of the photosynthetic apparatus was confirmed by respiration rate measurements, indicating a growth-phase dependent response to light. Comparative genomics with other members of the <it>Roseobacter </it>clade revealed several genomic regions that were only conserved in the two <it>Roseobacter </it>species. One of those regions encodes a variety of genes that might play a role in host association of the organisms. The catabolism of different carbon and nitrogen sources was predicted from the genome and combined with experimental data. In several cases, e.g. the degradation of some algal osmolytes and sugars, the genome-derived predictions of the metabolic pathways in <it>R. litoralis </it>differed from the phenotype.</p> <p>Conclusions</p> <p>The genomic differences between the two <it>Roseobacter </it>species are mainly due to lateral gene transfer and genomic rearrangements. Plasmid pRLO149_83 contains predominantly recently acquired genetic material whereas pRLO149_94 was probably translocated from the chromosome. Plasmid pRLO149_63 and one plasmid of <it>R. denitrifcans </it>(pTB2) seem to have a common ancestor and are important for cell envelope biosynthesis. Several new mechanisms of substrate degradation were indicated from the combination of experimental and genomic data. The photosynthetic activity of <it>R. litoralis </it>is probably regulated by nutrient availability.</p

Abstracts from the NIHR INVOLVE Conference 2017

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Ascorbic acid oxidase in barley and malt and its possible role during mashing

Ascorbic acid oxidase (AAO) develops in the embryo tissues of barley during steeping and initial stages of germination. Two AAO enzymes have been identified. One of them is of remarkably low molecular weight (&lt;10,000). Both are very heat tolerant and capable of acting over a broad pH range. Both enzymes would be expected to function during conversion temperatures of mashing. Indeed, addition of ascorbic acid to mashes results in the survival of higher levels of polyphenol and thiols into wort and a reduced color in that wort, commensurate with AAO preferentially consuming oxygen which, thus, is less readily available for other reactions in mashes, including thiol oxidation and polyphenol oxidation. © 2014 American Society of Brewing Chemists, Inc

Ascorbic acid oxidase in barley and malt and its possible role during mashing

Ascorbic acid oxidase (AAO) develops in the embryo tissues of barley during steeping and initial stages of germination. Two AAO enzymes have been identified. One of them is of remarkably low molecular weight (&lt;10,000). Both are very heat tolerant and capable of acting over a broad pH range. Both enzymes would be expected to function during conversion temperatures of mashing. Indeed, addition of ascorbic acid to mashes results in the survival of higher levels of polyphenol and thiols into wort and a reduced color in that wort, commensurate with AAO preferentially consuming oxygen which, thus, is less readily available for other reactions in mashes, including thiol oxidation and polyphenol oxidation. © 2014 American Society of Brewing Chemists, Inc