Genetic Polymorphisms in CYP2E1: Association with Schizophrenia Susceptibility and Risperidone Response in the Chinese Han Population

CYP2E1 is a member of the cytochrome P450 superfamily, which is involved in the metabolism and activation of both endobiotics and xenobiotics. The genetic polymorphisms of CYP2E1 gene (Chromosome 10q26.3, Accession Number NC_000010.10) are reported to be related to the development of several mental diseases and to be involved in the clinical efficacy of some psychiatric medications. We investigated the possible association of CYP2E1 polymorphisms with susceptibility to schizophrenia in the Chinese Han Population as well as the relationship with response to risperidone in schizophrenia patients.In a case-control study, we identified 11 polymorphisms in the 5' flanking region of CYP2E1 in 228 schizophrenia patients and 384 healthy controls of Chinese Han origin. From among the cases, we chose 130 patients who had undergone 8 weeks of risperidone monotherapy to examine the relationship between their response to risperidone and CYP2E1 polymorphisms. Clinical efficacy was assessed using the Brief Psychiatric Rating Scale (BPRS).Statistically significant differences in allele or genotype frequencies were found between cases and controls at rs8192766 (genotype p = 0.0048, permutation p = 0.0483) and rs2070673 (allele: p = 0.0018, permutation p = 0.0199, OR = 1.4528 95%CI = 1.1487-1.8374; genotype: p = 0.0020, permutation p = 0.0225). In addition, a GTCAC haplotype containing 5 SNPs (rs3813867, rs2031920, rs2031921, rs3813870 and rs2031922) was observed to be significantly associated with schizophrenia (p = 7.47E-12, permutation p<0.0001). However, no association was found between CYP2E1 polymorphisms/haplotypes and risperidone response.Our results suggest that CYP2E1 may be a potential risk gene for schizophrenia in the Chinese Han population. However, polymorphisms of the CYP2E1 gene may not contribute significantly to individual differences in the therapeutic efficacy of risperidone. Further studies in larger groups are warranted to confirm our results

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase&nbsp;1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation&nbsp;disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age&nbsp; 6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score&nbsp; 652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc&nbsp;= 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N&nbsp;= 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in&nbsp;Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in&nbsp;Asia&nbsp;and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Role of natural phenolics in hepatoprotection: A mechanistic review and analysis of regulatory network of associated genes

The liver is not only involved in metabolism and detoxification, but also participate in innate immune function and thus exposed to frequent target Thus, they are the frequent target of physical injury. Interestingly, liver has the unique ability to regenerate and completely recoup from most acute, non-iterative situation. However, multiple conditions, including viral hepatitis, non-alcoholic fatty liver disease, long term alcohol abuse and chronic use of medications can cause persistent injury in which regenerative capacity eventually becomes dysfunctional resulting in hepatic scaring and cirrhosis. Despite the recent therapeutic advances and significant development of modern medicine, hepatic diseases remain a health problem worldwide. Thus, the search for the new therapeutic agents to treat liver disease is still in demand. Many synthetic drugs have been demonstrated to be strong radical scavengers, but they are also carcinogenic and cause liver damage. Present day various hepatic problems are encountered with number of synthetic and plant based drugs. Nexavar (sorafenib) is a chemotherapeutic medication used to treat advanced renal cell carcinoma associated with several side effects. There are a few effective varieties of herbal preparation like Liv-52, silymarin and Stronger neomin phages (SNMC) against hepatic complications. Plants are the huge repository of bioactive secondary metabolites viz; phenol, flavonoid, alkaloid etc. In this review we will try to present exclusive study on phenolics with its mode of action mitigating liver associated complications. And also its future prospects as new drug lead

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Two Types of Functionally Distinct Fiber Containing Structural Protein Complexes Are Produced during Infection of Adenovirus Serotype 5

<div><p>Adenoviruses are common pathogens. The localization of their receptors coxsackievirus and adenovirus receptor, and desmoglein-2 in cell-cell junction complexes between polarized epithelial cells represents a major challenge for adenovirus infection from the apical surface. Structural proteins including hexon, penton base and fiber are excessively produced in serotype 5 adenovirus (Ad5)-infected cells. We have characterized the composition of structural protein complexes released from Ad5 infected cells and their capacity in remodeling cell-cell junction complexes. Using T84 cells as a model for polarized epithelium, we have studied the effect of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. The initial Ad5 infection in T84 cell culture was inefficient. However, progressive distortion of cell-cell junction in association with fiber release was evident during progression of Ad5 infection. Incubation of T84 cell cultures with virion-free supernatant from Ad5 infected culture resulted in distortion of cell-cell junctions and decreased infectivity of Ad5-GFP vector. We used gel filtration chromatography to fractionate fiber containing virion–free supernatant from Ad5 infected culture supernatant. Fiber containing fractions were further characterized for their capacity to inhibit the infection of Ad5-GFP vector, their composition in adenovirus structural proteins using western blot and LC-MS/MS and their capacity in remolding cell-cell junctions. Fiber molecules in complexes containing penton base and hexon, or mainly hexon were identified. Only the fiber complexes with relatively high content of penton base, but not the fiber-hexon complexes with low penton base, were able to penetrate into T84 cells and cause distortion of cell-cell junctions. Our findings suggest that these two types of fiber complexes may play different roles in adenoviral infection.</p></div

Remodeling of cell-cell junction between T84 epithelial cells by virion-free supernatant from Ad5 infected culture.

<p>Virion-free supernatant from Ad5-infected A549 culture at day 3 post infection with Ad5 at a MOI of 1 were prepared as conditioned media (CM) by ultra-centrifugation at 108 000 g. (A) T84 cells cultured on the insert of transwell until the development of TJ were apically treated with 100 μl Ad5 CM (corresponding to 4 functional units of fiber molecules) for 6 hr. CAR and DSG-2 were immuno-labeled with rabbit anti-CAR antibody and mouse anti-DSG-2 antibody followed by Alexa Flour 488 conjugated donkey anti-rabbit IgG and Alexa Flour 568 conjugated donkey-anti mouse IgG. The localization of CAR (green) and DSG-2 (red) in the control (ctrl, treated with supernatant from non-infected A549 cultures prepared in parallel to the CM from Ad5 infected A549 cultures) and Ad5 CM-treated T84 cultures are documented. Data in the XZ planes show the rearrangement of CAR and DSG2 localization. (B) T84 cells cultured as in (A) were basolaterally treated with Ad5 CM for 6 hr. Data on XY plane show diffuse CAR staining at cell-cell junction and cytoplasmic staining of CAR, as well as fiber staining following supernantant treatment; data on XZ plane show the apex-localization of CAR. Internalization of CAR and fiber molecules were found following either apical or basolateral application of Ad5 CM. Cells were visualized using Zeiss confocal microscopy LSM510. Bar: 10 μm.</p

Consequences of cell-cell junction remodeling on Ad infection.

<p>(A) T84 cultures with well-developed cell-cell junctions were pre-treated with Ad5 CM (corresponding to 4 functional units of fiber molecules) or with supernatant from mock infected culture from the apical surface for 6 hr, and subsequently infected with Ad5-GFP or Ad5F35-GFP at a MOI of 200 and 100, respectively. The infected cells were visualized by GFP at 48 hpi. Pre-treatment with Ad5 CM inhibited the infection of Ad5-GFP but promoted the infection of Ad5F35-GFP. Images were taken using an Olympus IX70 microscopy with a 20x objective. (B) Average fluorescent focus forming units (FFU) per slide of 6 individual photos as shown in (A). Each treatment was preformed in triplicate and 2 photos were taken from each trial. The FFU were analyzed and calculated by ImageJ program (n = 6, *: P < 0.05, ***: P < 0.001, <i>t</i>-test). The data shown are representative of three experiments performed.</p

Identification and characterization of structure protein complexes in the Ad5 CM.

<p>(A) Elution profile of gel filtration chromatography. One ml of concentrated Ad5 CM was separated by Superdex 200 column at a flow rate of 0.5 ml/min. Protein concentration of each fraction was monitored with absorbance at 280 nm (Y axis). The indicated fractions (arrow), named according to their positions on the collection rack, were selected for the analyses performed in panels B to E. (B) Upper panel: western-blot analysis for the detection of fiber (F) and penton base (PB) in the fractions from (A); lower panel: SimplyBlue staining in the corresponding SDS-PAGE gel. Fiber molecules were abundant in B12 and C5 fractions, penton base mainly in B12 fraction. (C) Identification of Ad structure proteins in fiber-containing fractions by cell surface binding assay. A549 cells were incubated with 200 μl of the indicated fractions for 15 min on ice. Cells were then washed and incubated with the 4D2 anti-fiber mAb in combination with rabbit anti-penton base antibody, followed by staining with Alexa Flour 647 conjugated goat anti-mouse IgG and Alexa Flour 488 conjugated goat anti-rabbit antibody. Parallel samples stained with 4D2 mAb in combination with FITC-conjugated goat anti-hexon antibody were then stained with Alexa Flour 647 conjugated goat anti-mouse IgG. The cells were analyzed in FACS Calibur for the double stained cells. The dot-plots shown are representative of three independent experiments. Fiber, penton base and hexon were detected in B12 fraction, whereas only hexon and fiber were detected in C5 fraction. (D) Characterization of structural protein complexes by native PAGE and western-blot analysis. Proteins in different fractions were separated by pre-casted 4–20% native PAGE gel and blotted onto PVDF membrane. The membrane was probed with anti-fiber, anti-penton base and anti-hexon antibodies simultaneously and then further probed with the secondary antibodies used in (C). The membrane was scanned by a PharosFX Plus system (Bio-Rad) using 3-laser channels for the visualization of specific binding bands for different antigens. SimplyBlue protein staining of the native PAGE gel (left panel) depicts that B12 and C5 fraction contain protein complexes of about 720 kDa and 440 kDa, respectively. The area on the gel corresponding to the western-blot data shown in the right panel is indicated by the rectangle. Fiber, hexon and penton base were detected in the B12 fraction, whereas fiber and hexon were detected in the C5 fraction. (E) Capacity of fiber containing fractions in inhibition of Ad5-GFP infection in A549 cells. The fractions containing fiber molecule (B10, B12, C5 and D7) were first concentrated by a factor of 5 and subsequently diluted as indicated with fresh cell culture media, 200 μl of each dilution were applied to each well of A549 cells cultured in 24-well plates for 30 min at 37°C. Following two washes with fresh media, cells were infected with Ad5-GFP virus at a MOI of 100 for 1 hr at 37°C. The percentage of infected cells was analyzed with FACS assay as GFP positive cells at 24 hpi. NC: negative control samples (mock infection); PC: positive control samples (infection without pre-treatment with Ad5 CM).</p

Fiber is released and TJs exhibit progressive distortion over time after Ad5 infection of polarized T84 cultures from the apical surface.

<p>(A) Fiber molecules were already released prior to cell lysis from infected cells. T84 cells cultured on transwell inserts with well-developed TJs were infected with Ad5 at a MOI of 20 pfu/cell from the apical surface. Cultures were washed twice with the DMEM-F12 medium at 4 hrs post infection. Merged and separated images of hexon (green) and fiber (red) staining show that at 30 hpi, fiber molecules were released from infected cells positively stained with hexon and bound to non-infected bystander cells. Bar: 20 μm. (B) Progressive distortion of TJs. At 30 hpi, CAR staining (green) was predominantly localized within distinct TJs at the apex of T84 cell layer. At 60 hpi, distorted staining pattern of CAR was detected at the apex, basolateral membrane and in cytoplasma of T84 cells. Internalized fiber molecules (red dots, arrow heads) and co-localization of internalized fiber and CAR (yellow dots, arrows) in non-infected cells are indicated. Bar: 10 μm. Cells were visualized using Zeiss confocal microscopy LSM510. Images are representative of multiple experiments performed in this project. The crosshairs in the plots indicate the positions for the XZ plane.</p