Human dendritic cells and hepatitis C Virus

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers. Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported. To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients’ blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection. Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system. The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients

The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells

Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22 resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders. Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM

The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells

Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22 resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders. Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM

Reductions in circulating levels of IL-16, IL-7 and VEGF-A in myalgic encephalomyelitis/chronic fatigue syndrome

Recently, differences in the levels of various chemokines and cytokines were reported in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) as compared with controls. Moreover, the analyte profile differed between chronic ME/CFS patients of long duration versus patients with disease of less than 3 years. In the current study, we measured the plasma levels of 34 cytokines, chemokines and growth factors in 100 chronic ME/CFS patients of long duration and in 79 gender and age-matched controls. We observed highly significant reductions in the concentration of circulating interleukin (IL)-16, IL-7, and Vascular Endothelial Growth Factor A (VEGF-A) in ME/CFS patients. All three biomarkers were significantly correlated in a multivariate cluster analysis. In addition, we identified significant reductions in the concentrations of fractalkine (CX3CL1) and monokine-induced-by-IFN-γ (MIG; CXCL9) along with increases in the concentrations of eotaxin 2 (CCL24) in ME/CFS patients. Our data recapitulates previous data from another USA ME/CFS cohort in which circulating levels of IL-7 were reduced. Also, a reduced level of VEGF-A was reported previously in sera of patients with Gulf War Illness as well as in cerebral spinal fluid samples from a different cohort of USA ME/CFS patients. To our knowledge, we are the first to test for levels of IL-16 in ME/CFS patients. In combination with previous data, our work suggests that the clustered reduction of IL-7, IL-16 and VEGF-A may have physiological relevance to ME/CFS disease. This profile is ME/CFS-specific since measurement of the same analytes present in chronic infectious and autoimmune liver diseases, where persistent fatigue is also a major symptom, failed to demonstrate the same changes. Further studies of other ME/CFS and overlapping disease cohorts are warranted in future

Enhanced activation of memory, but not naive, B cells in chronic hepatitis C virus-infected patients with cryoglobulinemia and advanced liver fibrosis.

Mixed cryoglobulinemia is the most common extrahepatic disease manifestation of chronic hepatitis C virus (HCV) infection, where immunoglobulins precipitate at low temperatures and cause symptoms such as vasculitis, glomerulonephritis and arthralgia. HCV-associated cryoglobulinemia is also strongly linked with the development of B cell non-Hodgkin lymphoma. Abnormal B cell function in HCV infections can lead to the formation of HCV cryoglobulin complexes that usually comprise monoclonal rheumatoid factor and HCV-specific immune complexes. The aim of this study was to characterize the activation phenotype of B cells from patients with chronic HCV infection in comparison to healthy controls using flow cytometry. In addition, we determined how the activation status varies depending on the presence of cryoglobulinemia and advanced liver fibrosis. We found that only memory B cells, not naïve cells, were significantly activated in chronic HCV infection when compared with healthy controls. We also identified markers of memory B cell activation that were specific for HCV patients with cryoglobulinemia (CD86, CD71, HLA-DR) and advanced liver disease (CD86). Our results demonstrate that HCV infection has differential effects on B cells depending on the severity of hepatic and extrahepatic disease

Memory, but not naïve B cells are significantly activated in chronically infected HCV patients.

<p>PBMCs were freshly isolated and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068308#pone-0068308-g002" target="_blank">Figure 2</a>. Antibodies to CD183, CD71, CD86, CD69, HLA-DR and CD40 were added in combinations with CD19 and CD27 to gate on memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. The percent positive was calculated based on mouse isotype controls and the geometric mean fluorescent intensities (MFI) for each marker were determined. HLA-DR and CD40 are expressed by all B cells therefore only the MFI is shown. Horizontal lines on graphs represent median values. For CD71 analysis, only 48 HCV and 47 healthy control samples were stained. ***, P<0.001, **, P<0.01, *, P<0.05, n.s., not significant.</p

B cell subset frequencies are unchanged in HCV patients compared to controls.

<p>(<b>A–C</b>) 7.5×10⁵ freshly isolated PBMCs from chronically infected HCV patients (n = 54) or healthy controls (n = 50) were incubated with fluorescently labeled antibodies to CD19, CD27 and CD5 for multi-color flow cytometry analysis. (<b>A</b>) The total percent of CD19+ B cells within the lymphocyte gate and number of B cells per ml of blood based on our isolations. (<b>B</b>) The percent of memory (CD19+CD27+) and naïve (CD19+CD27−) and number of each subset per ml of blood. <b>(C)</b> The percent and number per ml of CD5+CD19+ B cells. Horizontal lines on graphs represent means +/− SEM (percentages) or median (number of cells/ml) values. In (C) only 48 HCV and 47 healthy control samples were tested for CD5. *, P<0.05; n.s., not significant.</p

HCV patients with advanced fibrosis have increased expression of CD86 on memory B cells.

<p>Fibrosis scores were determined as outlined in the Materials and Methods, where scores of 3–4 are considered advanced fibrosis/cirrhosis (F4). PBMCs were freshly isolated and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068308#pone-0068308-g003" target="_blank">Figure 3</a>. The expression level and percent positive of cells expressing CD86 (geometric mean fluorescent intensity (MFI) or percent positive compared to mouse isotype controls) were calculated for memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. Horizontal lines represent median values. *, P<0.05; n.s., not significant.</p

Clinical characteristics of HCV+ patients and healthy controls in study.

<p>Abbreviations: N/A, not applicable, ALT, alanine aminotransferase (normal values = <50), GGT, gamma-glutamyl transpeptidase (normal levels = <70 (males), <55 (females)), IU, International units, IFN, interferon.</p>*<p>determined from biopsy or Fibroscan.</p

Gating strategy for flow cytometry analysis of memory and naïve B cell activation.

<p>Lymphocytes were gated by FSC/SSC properties and memory B cells (CD19+CD27+) and naïve B cells (CD19+CD27−) were analyzed within the lymphocyte gate for the expression of 6 different markers described in the Materials and Methods. Histograms show a representative example of the expression of CD183 (numbers represent geometric mean fluorescent intensity) on memory and naïve B cells from a healthy control (HCV-) and chronic HCV patient (HCV+).</p