Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Modulating the strength and threshold of NOTCH oncogenic signals by mir-181a-1/b-1.

Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

Primary biliary cirrhosis is associated with altered hepatic microRNA expression.

MicroRNAs (miRNAs) are small RNA molecules that negatively regulate protein coding gene expression and are thought to play a critical role in many biological processes. Aberrant levels of miRNAs have been associated with numerous diseases and cancers, and as such, miRNAs have gain much interests as diagnostic biomarkers, and as therapeutic targets. However, their role in autoimmunity is largely unknown. The aims of this study are to: (1) identify differentially expressed miRNAs in human primary biliary cirrhosis (PBC); (2) validate these independently; and (3) identify potential targets of differentially expressed miRNAs. We compared the expression of 377 miRNAs in explanted livers form subjects with PBC versus controls with normal liver histology. A total of 35 independent miRNAs were found to be differentially expressed in PBC (p < 0.001). Quantitative PCR was employed to validate down-regulation of microRNA-122a (miR-122a) and miR-26a and the increased expression of miR-328 and miR-299-5p. The predicted targets of these miRNAs are known to affect cell proliferation, apoptosis, inflammation, oxidative stress, and metabolism. Our data are the first to demonstrate that PBC is characterized by altered expression of hepatic miRNA; however additional studies are required to demonstrate a causal link between those miRNA and the development of PBC.

Dissecting microRNA-mediated gene regulation and function in T-cell development.

MicroRNAs (miRNAs) are abundant approximately 22-nucleotide regulatory RNAs encoded in animal genomes. They are thought to exhibit diverse biological functions in animals by targeting messenger RNAs (mRNAs) for degradation or translational repression. Here we use T-cell development as a model to illustrate methods and strategies for dissecting the post transcriptional gene regulatory networks controlled by miRNAs and their roles in the differentiation of T-cell precursors. The process involves the identification of miRNA genes in rare T-cell progenitors, determining miRNA expression during T-cell development, characterizing miRNA function in T-cell development using an in vitro assay, and identifying functionally relevant gene(s) regulated by miRNAs.

Elevated expression of tyrosine kinase DDR2 in primary biliary cirrhosis.

BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and cholestasis, leading to fibrosis. METHODS: We utilized a novel restriction analysis system to profile the expression of tyrosine kinases (TKs). This methodology targets a conserved sequence present in the majority of human TKs, and exploits the known restriction map of the TK cDNA sequences. We isolated mRNA from biliary epithelial cell (BEC)-enriched cell fractions, amplified the TK transcripts using degenerative primers, and identified specific TKs by restriction enzyme digest analysis and then performed in situ hybridization. RESULTS: BEC-enriched samples from PBC livers displayed marked expression of discoidin domain receptor-2 (DDR2), whereas, non-diseased livers showed no detectable DDR2. Furthermore, in situ hybridization of PBC livers revealed that DDR2 is expressed in the small bile duct epithelial regions as well as in fibroblasts/stromal cells of fibrotic regions. A similar pattern was observed in livers of primary sclerosing cholangitis (PSC), although the amount of small ducts that were positively stained was lower than in PBC. Furthermore, cirrhotic livers of patients with other diseases, including alcoholic liver disease and chronic hepatitis C, DDR2 transcripts were noted only within fibrotic lesions and the degree of intensity was much lower than in PBC and PSC. CONCLUSIONS: DDR2, a TK that is stimulated by fibrillar collagens that accumulate in cirrhotic livers, is present at elevated levels in the small bile ducts of PBC patients. DDR2 is part of a positive feedback loop in which its enhanced expression leads to enhanced deposition of fibrillar collagens (types I andIII). These fibrillar collagens can also provide binding sites for immune mediators, such as cytokines and chemokines. Therefore, unusually high DDR2 expression in the bile ducts of PBC patients could contribute to duct injury by altering local cytokine levels and thereby increasing immune-mediated damage.

Impaired indoleamine 2,3-dioxygenase production contributes to the development of autoimmunity in primary biliary cirrhosis.

The immunomodulatory effects of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) have been elucidated at a cellular level and implicated in the pathogenesis of several complex diseases. Defects within the regulatory T cell compartment are one of the characteristics of primary biliary cirrhosis (PBC), an autoimmune chronic cholestatic liver disease, a phenotype that has also been shown in disease-mimicking animal models of this disease. We hypothesized that IDO dysregulation could lead to altered frequency and/or function of T cells at the level of antigen processing/presentation and we thus investigated IDO in peripheral monocytes and bile duct cells from patients with PBC. Both expression and activation manifested an impaired IFN-gamma response in peripheral monocytes while a peculiar IDO expression profile in bile duct cells characterized early stage PBC. Further, we observed an increased frequency of a gain-of-function SNP within the TGF-beta promoter region, a molecule known to suppress IDO transcription. In conclusion, we submit that an impaired IDO induction characterizes PBC and might represent a contributing factor in disease pathogenesis in association with several specific defects in the target tissue.

The anti-inflammatory properties of cocoa flavanols.

Signs of chronic or acute inflammation have been demonstrated in most cardiovascular diseases of multifactorial pathogenesis, including atherosclerosis and chronic heart failure. The triggers and mechanisms leading to inflammation may vary between clinical conditions but they share many common mediators, including specific patterns of eicosanoid and cytokine production. Certain cocoa-based products can be rich in a subclass of flavonoids known as flavanols, some of which have been found in model systems to possess potential anti-inflammatory activity relevant to cardiovascular health. Indeed, experimental evidence demonstrates that some cocoa-derived flavanols can reduce the production and effect of pro-inflammatory mediators either directly or by acting on signaling pathways. However, it should be noted that the evidence for any beneficial effects of cocoa flavanols in providing a meaningful anti-inflammatory action has been gathered predominantly from in vitro experiments. Therefore, additional research in well-designed human clinical experiments, using cocoa properly characterized in terms of flavanol content, would be a welcome addition to the evidence base to determine unambiguously if this benefit does indeed exist. If so, then flavanol-rich cocoa could be a potential candidate for the treatment, or possibly prevention, of the broad array of chronic diseases that are linked to dysfunctional inflammatory responses.

Altered monocyte responses to defined TLR ligands in patients with primary biliary cirrhosis.

The role of the adaptive immune response, with regard to the development of autoantibodies, has been extensively studied in primary biliary cirrhosis (PBC). However, the importance of innate immunity has been noted only recently. Based on the proposed role of microorganisms in the pathogenesis of the disease, we hypothesize that patients with PBC possess a hyper-responsive innate immune system to pathogen-associated stimuli that may facilitate the loss of tolerance. To address this issue, we isolated peripheral blood monocytes from 33 patients with PBC and 26 age-matched healthy controls and stimulated such cells in vitro with defined ligands for toll-like receptor (TLR) 2 (lipoteichoic acid; LTA), TLR3 (polyIC), TLR4 (lipopolysaccharide; LPS), TLR5 (flagellin), and TLR9 (CpG-B). Supernatant fluids from the cultures were analyzed for levels of 5 different pro-inflammatory cytokines, interleukin (IL)-1beta, IL-6, IL-8, IL-12p70, and TNF-alpha. After in vitro challenge with TLR ligands, PBC monocytes produced higher relative levels of pro-inflammatory cytokines, particularly IL-1beta, IL-6, IL-8, and TNF-alpha, compared with controls. In conclusion, monocytes from patients with PBC appear more sensitive to signaling via select TLRs, resulting in secretion of selective pro-inflammatory cytokines integral to the inflammatory response that may be critical in the breakdown of self-tolerance.

Genetic polymorphisms influencing xenobiotic metabolism and transport in patients with primary biliary cirrhosis.

Epidemiological data suggest that environmental factors may trigger autoimmunity in genetically susceptible individuals. In primary biliary cirrhosis (PBC), it has been postulated that halogenated xenobiotics can modify self-molecules, facilitating the breakdown of tolerance to mitochondrial antigens. The transport and metabolism of xenobiotics is highly dependent on key genetic polymorphisms that alter enzymatic phenotype. We analyzed genomic DNA from 169 patients with PBC and 225 geographically and sex-matched healthy subjects for polymorphisms of genes coding for cytochromes P450 (CYPs) 2D6 (CYP2D6*4, CYP2D6*3, CYP2D6*5, and CYP2D6*6) and 2E1 (cl/c2), multidrug resistance 1 (MDR1 C3435T) P-glycoprotein, and pregnane X receptor (PXR C-25385T, C8055T, and A7635G). We compared the genotype frequencies in patients and controls and also correlated polymorphisms with PBC severity. The distributions of the studied genotypes did not significantly differ between patients and controls. However, when clinical characteristics of patients with PBC were compared according to genotype, the CYP2E1 c2 allele was associated with signs of more severe disease. In conclusion, genetic polymorphisms of CYP 2D6 and 2E1, PXR, and MDR1 do not appear to play a role in the onset of PBC.

Sidechain biology and the immunogenicity of PDC-E2, the major autoantigen of primary biliary cirrhosis.

The E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2) is the immunodominant autoantigen of primary biliary cirrhosis. Whereas lipoylation of PDC-E2 is essential for enzymatic activity and predominates under normal conditions, other biochemical systems exist that also target the lysine residue, including acylation of fatty acids or xenobiotics and ubiquitinylation. More importantly, the immunogenicity can be affected by derivatization of the lysine residue, as the recognition of lipoylated PDC-E2 by patient autoantibodies is enhanced compared with octanoylated PDC-E2. Furthermore, our laboratory has shown that various xenobiotic modifications of a peptide representing the immunodominant region of PDC-E2 are immunoreactive against patient sera. The only purported regulatory system that prevents the accumulation of potentially autoreactive PDC-E2 is glutathionylation, in which the lysine-lipoic acid moiety is further modified with glutathione during apoptosis. Interestingly, this system is found in several cell lines, including HeLa, Jurkat, and Caco-2 cells, but not in cholangiocytes and salivary gland epithelial cells, both of which are targets for destruction in primary biliary cirrhosis. Hence, the failure of this or other regulatory system(s) may overwhelm the immune system with immunogenic PDC-E2 that can initiate the breakdown of tolerance in a genetically susceptible individual. In this review the authors survey the data available on the biochemical life of PDC-E2, with particular emphasis on the lysine residue and its known interactions with machinery involved in various posttranslational modifications.

Effects of a Combination of Traditional Chinese Botanicals (Immune+) on the Secretion of Interleukin-1beta and Interferon-gamma by Peripheral Blood Mononuclear Cells.

The use of herbal and other botanical products, including those used extensively in traditional Chinese medicine, has increased dramatically in the last decade. Yet, little scientific research exists concerning their efficacy and safety. We examined the effects of Immune+, a combination of five botanicals frequently used in traditional Chinese medicine, on the production of two cytokines. For this purpose, unstimulated or phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells from healthy volunteers were incubated with different concentrations of Immune+. The secretion of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) was measured after 72 hours of incubation. At the highest concentration tested (100 micro g/ml), Immune+ significantly increased the secretion of IL-1beta. Importantly, PHA alone had no effect on IL-1beta production, and the combination of PHA with Immune+ resulted in the same increase in IL-1beta production as seen with the botanical extract alone. Immune+ did not have any detectable effect on either unstimulated or PHA-stimulated IFN-gamma synthesis. These in vitro data support the concept that Immune+ may enhance human immune responses.