a-protocol-for-analyzing-hepatitis-c-virus-replication

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Cedars-Sinai Medical Center

Control of 125-dihydroxyvitamin D (1,25-(OH)2D) synthesis is believed to be primarily at the level of expression of the vitamin D-1-hydroxylase (CYP1alpha; CYP1alpha) gene. Once transcribed, generation of product, as catalyzed by 1-hydroxylase, depends upon the availability of various co-factors, molecular oxygen, electrons as well as substrate to the enzyme. Here we provide evidence that the quantity of product 1,25-(OH)2D generated also relies on the presence and level of expression of the intracellular vitamin D binding protein-1 (IDBP-1) and its capacity to promote 24-hydroxylase (CYP24) gene expression. Stable transfection of the IDBP-1 cDNA increased 1,25-(OH)2D synthesis up to 700% (p < 0.001) in cells devoid of 24-hydroxylating potential but only 70% (p = 0.018) in cells in which the CYP24 gene is expressed. IDBP-1-mediated increase in 1,25-(OH)2D production was independent of any change in CYP1alpha expression but highly dependent on the ability of exogenously-added or endogenously-synthesized 1,25-(OH)2D to stimulate CYP24 gene expression. These data suggest that IDBP-1 is capable of controlling 1,25-(OH)2D production by modulating the delivery of 1) substrate 25-OHD to in the mitochondrial CYP1alpha gene product and 2) CYP1alpha product 1,25-(OH)2D to the vitamin D receptor for upregulation of expression of the catabolic CYP24 gene.

Regulation of 1,25-dihydroxyvitamin d synthesis by intracellular vitamin d binding protein-1.

Using vitamin D-resistant New World primates as model of natural diversity for sterol/steroid action and metabolism, two families of novel intracellular vitamin D regulatory proteins have been discovered and their human homologs elucidated. The first family of proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), initially considered to function only as pre-mRNA-interacting proteins, have been demonstrated to be potent cis-acting, trans-dominant regulators of vitamin D hormone-driven gene transactivation. The second group of proteins bind 25-hydroxylated vitamin D metabolites. Their overexpression increases vitamin D receptor (VDR)-directed target gene expression. We found that these intracellular vitamin D binding proteins (IDBPs) are homologous to proteins in the heat shock protein-70 family. Our ongoing studies indicate directly or indirectly through a series of protein interactions that the IDBPs interact with hydroxylated vitamin D metabolites and facilitate their intracellular targeting.

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism.

Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease.

Alternative splicing of vitamin D-24-hydroxylase: a novel mechanism for the regulation of extrarenal 1,25-dihydroxyvitamin D synthesis.

Tissue availability of the active vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] is dependent on expression of the activating enzyme 1alpha-hydroxylase (CYP27b1) and its catabolic counterpart 24-hydroxylase (CYP24). The activity of these two enzymes is in turn controlled by factors including affinity of the serum vitamin D-binding protein (DBP) for 25-hydroxyvitamin D [25(OH)D]; the availability of enzyme cofactors; and the relative amount of hydroxylase gene product expressed. In recent years, it has become clear that directed trafficking of substrate and enzyme is also a pivotal component of the regulated process of hormone synthesis by both renal and extrarenal tissues expressing the CYP27b1 and CYP24 genes. Extracellular regulatory trafficking events are defined by the quantity of substrate 25(OH)D entering the circulatory pool. Entry into some target cells in vivo, such as the macrophage and proximal renal tubular epithelial cells, requires 25(OH)D binding to serum DBP, followed by recognition, internalization, and intracellular release. The "released" intracellular substrate is moved to specific intracellular destinations (i.e., the mitochondrial CYP enzymes and the vitamin D receptor [VDR]) by the hsc70 family of chaperone proteins. Synthesis of 1,25(OH)(2)D is also regulated by CYP24 and its metabolically inactive splice variant CYP24-SV. Finally, initiation of transcription of 1,25(OH)(2)D-regulated genes, such as the CYP24, requires movement of the CYP27b1 product, 1,25(OH)(2)D, to the VDR in the same cell for intracrine action or export to another cell for paracrine action. In either case, the 1,25(OH)(2)D ligand is required for the VDR to heterodimerize with the retinoid x receptor and compete away the dominant-negative acting, heterogeneous nuclear ribonucleoprotein (hnRNP)-related, vitamin D response element-binding proteins that inhibit hormone-directed transactivation of genes. In this review, we use vitamin D-directed events in the human innate immune response to Mycobacterium tuberculosis as a physiologically relevant model system in which to highlight the importance of these intracellular traffic patterns.

Substrate and enzyme trafficking as a means of regulating 1,25-dihydroxyvitamin D synthesis and action: the human innate immune response.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, [1,25(OH)2D3] has potent actions on innate and adaptive immunity. Although endocrine synthesis of 1,25(OH)2D3 takes place in the kidney, the enzyme that catalyzes this, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27b1 in humans, Cyp27b1 in mice), is expressed at many extra-renal sites including the colon. We have shown previously that colonic expression of CYP27b1 may act to protect against the onset of colitis. To investigate this further, we firstly characterized changes in Cyp27b1 expression in a mouse model of colitis. Mice treated with dextran sodium sulfate (DSS) showed weight loss, histological evidence of colitis, and increased expression of inflammatory cytokines. This was associated with decreased renal expression of Cyp27b1 (5-fold, P=0.013) and lower serum 1,25(OH)2D3 (51.8+/-5.9 pg/nl vs. 65.1+/-1.6 in controls, P<0.001). However, expression of CYP27b1 was increased in the proximal colon of DSS mice (4-fold compared with controls, P<0.001). Further studies were carried out using Cyp27b1 null (-/-) mice. Compared with+/-controls the Cyp27b1-/-mice showed increased weight loss (4.9% vs. 22.8%, P<0.001) and colitis. This was associated with raised IL-1 in the distal colon and IL-17 in the proximal and distal colon. Conversely, DSS-treated Cyp27b1-/-mice exhibited lower IL-10 in the proximal colon and toll-like receptors 2 and 4 in the distal colon. These data indicate that both local and endocrine synthesis of 1,25(OH)2D3 affect colitis in DSS-treated mice. Lack of Cyp27b1 exacerbates disease in this model, suggesting that similar effects may occur with vitamin D deficiency.

Altered endocrine and autocrine metabolism of vitamin D in a mouse model of gastrointestinal inflammation.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

Vitamin d-directed rheostatic regulation of monocyte antibacterial responses.

Riportiamo qui una piattaforma di imaging multispettrale non invasiva per il monitoraggio di immagini di fluorescenza e riflettanza spettrale da carcinoma mammario primario e linfonodi metastatici nel modello di ratto preclinica in vivo. Il sistema &egrave; costruito intorno a una sorgente di luce del monocromatore e un filtro sintonizzabile acusto-ottico (AOTF) per selezione spettrale. Analisi quantitativa dei profili di riflettanza misurato in presenza di un colorante lymphazurin ampiamente utilizzato dimostra chiaramente la capacit&agrave; della piattaforma di imaging proposta di rilevare le firme spettrali tumore-associati in tumori primari cos&igrave; come i vasi linfatici metastatici. Cambiamenti associati al tumore vascolare ossigenazione e pressione del fluido interstiziale sono motivate le fonti fisiologica dei profili di riflettanza misurato. Discutiamo anche il potenziale traslazionale della nostra piattaforma di imaging in ambiente clinico in fase intraoperatoria.

Non invasivi, contrasto migliorato Spectral Imaging del seno cancro firme in modelli animali preclinici In vivo.

Nous rapportons ici une plateforme d&#39;imagerie multispectrale non invasive pour surveiller les images spectrales de r&eacute;flectance et de fluorescence de carcinome mammaire primaire et des ganglions m&eacute;tastatiques dans mod&egrave;le pr&eacute;clinique de rat in vivo. Le syst&egrave;me est construit autour d&#39;une source lumineuse de monochromateur et un filtre accordable acousto-optique (AOTF) pour la s&eacute;lection spectrale. Analyse quantitative des profils r&eacute;flectance mesur&eacute;e en pr&eacute;sence d&#39;un colorant lymphazurin r&eacute;pandu d&eacute;montre clairement la capacit&eacute; de la plate-forme d&#39;imagerie propos&eacute;e pour d&eacute;tecter des signatures spectrales associ&eacute;es aux tumeurs dans les tumeurs primaires, mais aussi les vaisseaux lymphatiques m&eacute;tastatiques. Modifications associ&eacute;es &agrave; la tumeur en oxyg&eacute;nation vasculaire et la pression du fluide interstitielle sont motiv&eacute;es les sources physiologiques des profils mesur&eacute;s de r&eacute;flectance. Nous discutons &eacute;galement le potentiel translationnel de notre plate-forme d&#39;imagerie en milieu clinique perop&eacute;ratoire.

Non invasifs, contraste accentu&eacute; Imaging de Breast Cancer signature spectrale en pr&eacute;clinique Animal mod&egrave;les In vivo.

We report here a non-invasive multispectral imaging platform for monitoring spectral reflectance and fluorescence images from primary breast carcinoma and metastatic lymph nodes in preclinical rat model in vivo. The system is built around a monochromator light source and an acousto-optic tunable filter (AOTF) for spectral selection. Quantitative analysis of the measured reflectance profiles in the presence of a widely-used lymphazurin dye clearly demonstrates the capability of the proposed imaging platform to detect tumor-associated spectral signatures in the primary tumors as well as metastatic lymphatics. Tumor-associated changes in vascular oxygenation and interstitial fluid pressure are reasoned to be the physiological sources of the measured reflectance profiles. We also discuss the translational potential of our imaging platform in intra-operative clinical setting.

Non-invasive, Contrast-enhanced Spectral Imaging of Breast Cancer Signatures in Preclinical Animal Models In vivo.

Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.

Songyang Ren

Liver Program at Regenerative Medicine Institute,
Department of Biomedical Sciences,
Department of Surgery,
Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery

Cedars-Sinai Medical Center

A Protocol for Analyzing Hepatitis C Virus Replication

Songyang Ren

Liver Program at Regenerative Medicine Institute,Department of Biomedical Sciences,Department of Surgery,Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery

Cedars-Sinai Medical Center

A Protocol for Analyzing Hepatitis C Virus Replication

Liver Program at Regenerative Medicine Institute,
Department of Biomedical Sciences,
Department of Surgery,
Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery