A Fibrosis-Independent Hepatic Transcriptomic Signature Identifies Drivers of Disease Progression in Primary Sclerosing Cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a heterogeneous cholangiopathy characterized by progressive biliary fibrosis. RNA sequencing of liver tissue from patients with PSC (n = 74) enrolled in a 96-week clinical trial was performed to identify associations between biological pathways that were independent of fibrosis and clinical events. APPROACH AND RESULTS: The effect of fibrosis was subtracted from gene expression using a computational approach. The fibrosis-adjusted gene expression patterns were associated with time to first PSC-related clinical event (e.g., cholangitis, hepatic decompensation), and differential expression based on risk groups and Ingenuity Pathway Analysis were performed. Baseline demographic data were representative of PSC: median age 48 years, 71% male, 49% with inflammatory bowel disease, and 44% with bridging fibrosis or cirrhosis. The first principle component (PC1) of RNA-sequencing data accounted for 18% of variance and correlated with fibrosis stage (ρ = -0.80; P < 0.001). After removing the effect of fibrosis-related genes, the first principle component was not associated with fibrosis (ρ = -0.19; P = 0.11), and a semisupervised clustering approach identified two distinct patient clusters with differential risk of time to first PSC-related event (P < 0.0001). The two groups had similar fibrosis stage, hepatic collagen content, and α-smooth muscle actin expression by morphometry, Enhanced Liver Fibrosis score, and serum liver biochemistry, bile acids, and IL-8 (all P > 0.05). The top pathways identified by Ingenuity Pathway Analysis were eukaryotic translation inhibition factor 2 (eIF2) signaling and regulation of eIF4/p70S6K signaling. Genes involved in the unfolded protein response, activating transcription factor 6 (ATF6) and eIF2, were differentially expressed between the PSC clusters (down-regulated in the high-risk group by log-fold changes of -0.18 [P = 0.02] and -0.16 [P = 0.02], respectively). Clinical events were enriched in the high-risk versus low-risk group (38% [12/32] vs. 2.4% [1/42], P < 0.0001). CONCLUSIONS: Removing the contribution of fibrosis-related pathways uncovered alterations in the unfolded protein response, which were associated with liver-related complications in PSC.

In silico identification of RBD subdomain of spike protein from Pro322-Thr581 for applications in vaccine development against SARS-CoV2.

The three-dimensional hybrid structures of coronavirus spike proteins including the C-terminal sequence and receptor binding motif (RBM) was remodeled and energy minimized. Further, protein-protein docking show that Receptor Binding Domain (RBD) of SARS-CoV 2 Lys457-Pro490 bind on the surface of ACE2 receptor near N-terminal helices to form host-pathogen attachment. In this binding interface, SARS-CoV 2 shows a tight network of hydrogen bonds than other spike proteins from BtRsRaTG13-CoV, SARS-CoV, BtRsBeta-CoV, BtRsCoV-related, Pangolin-CoV (PCoV), human-CoV (hCoV), MERS-CoV (MCoV), Avian-CoV (ACoV) and PEDV1-CoV. Further studies show that subdomains from SARS-CoV 2 RBD Pro322-Thr581, SARS-CoV RBD Pro309-Pro575, BtRsRaTG13 RBD Thr581-Thr323, BtRsBeta-CoV RBD Ser311-Thr568, BtRsCoV-related Arg306-Pro575 and PCoV RBD Gln319-Ser589 show binding conformations with ACE2 like their full-length structures of spike proteins. In addition, the subdomains MCoV RBD Gly372-Val616, ACoV RBD Gly372-Val616 and PEDV1-CoV RBD Ala315-Tyr675 also binds on the surface of ACE2 similar to their full-length spike proteins. The B-Cell epitope mapping also identified main antigenic determinants predicting that these nine subdomains are highly useful in recombinant vaccine development in inducing cross neutralizing antibodies against SARS-CoV 2 spike protein and inhibits its attachment with ACE2.

Discovery of anti-influenza nucleoside triphosphates targeting the catalytic site of A/PR/8/34/H1N1 polymerase.

In an effort to develop potent anti-influenza drugs that inhibit the activity of influenza virus RNA-dependent RNA polymerase (IAV RdRp), a database of nucleoside triphosphates with ~800 molecules were docked with the homology model of IAV RdRp from A/PR/8/34/H1N1 strain. Out of top 12 molecules that bind with higher affinities to the catalytic site of IAV RdRp above and below the PB1 priming loop, only seven molecules decreased the transcriptional activity of the viral RNA polymerase with an IC50 in the range of 0.09-3.58 µM. Molecular docking combining with experimental study indicated that the molecules with linear chain are more effective in inhibiting IAV RdRp replication than the molecules with V-shaped and are cyclic in nature. A correlation between ΔG and LogIC50 for these seven compounds resulted an R 2 value of 0.73. Overall, these newly developed seven nucleoside triphosphates lay a strong foundation for the future development of a new therapeutics that can satisfy the Lipinski's rule of five exhibiting high specificity to the catalytic site of influenza-A viruses.

Defining a standard and weighted mathematical index for maturation of dendritic cells.

The concept of dendritic cell (DC) maturation generally refers to the changes in morphology and function of DCs. Conventionally, DC maturity is based on three criteria: loss of endocytic ability, gain of high-level capacity to present antigens and induce proliferation of T cells, and mobility of DCs toward high concentrations of CCL19. Impairment of DC maturation has been suggested as the main reason for infectivity or chronicity of several infectious agents. In the case of hepatitis C virus, this has been a matter of controversy for the last two decades. However, insufficient attention has been paid to the method of ex vivo maturation as the possible source of such controversies. We previously reported striking differences between DCs matured with different methods, so we propose the use of a standard quantitative index to determine the level of maturity in DCs as an approach to compare results from different studies. We designed and formulated a mathematically calculated index to numerically define the level of maturity based on experimental data from ex vivo assays. This introduces a standard maturation index (SMI) and weighted maturation index (WMI) based on strictly standardized mean differences between different methods of generating mature DCs. By calculating an SMI and a WMI, numerical values were assigned to the level of maturity achieved by DCs matured with different methods. SMI and WMI could be used as a standard tool to compare diversely generated mature DCs and so better interpret outcomes of ex vivo and in vivo studies with mature DCs.

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.

Prion 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 ∼35µM and 20µM.

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 3years. 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.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1.

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer.

Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.

Differential serum levels of eosinophilic eotaxins in primary sclerosing cholangitis, primary biliary cirrhosis, and autoimmune hepatitis.

To investigate pathogenic mechanisms of primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH), serum levels of 26 chemokines and cytokines were determined and compared with patients with chronic hepatitis C or in healthy controls. The chemokine eotaxin-3 (E3; CCL26), which recruits eosinophils to sites of inflammation, was found to be highly elevated in all PSC, PBC, and AIH patients compared with HCV patients and healthy controls. Eotaxin-1 (E1; CCL11), another eosinophil-specific chemokine, was elevated in PSC but reduced in PBC and AIH, while the macrophage-derived chemokine (MDC; CCL22) was lower in all PSC, PBC, and AIH patients compared with HCV patients and controls. By incorporating levels of the interleukin (IL)-15 into a diagnostic algorithm, PSC, PBC, and AIH patients could each be differentiated with good sensitivity and specificity. These findings represent the first study to compare the level of serum cytokine/chemokine levels among these related autoimmune-like liver diseases. Furthermore, our data indicate that the measurement of serum E3, E1, CCL22, and IL-15 levels can aid in the diagnosis of these clinically challenging diseases and shed light on the potential pathogenic mechanisms underlying these diseases. By suggesting a potential role for an allergic phenomenon involving eosinophils, which may define them as liver-specific allergic diseases, this may open up potential new therapeutic avenues by abrogating the action of these disease-associated immune modulators.

Progress towards a hepatitis C virus vaccine.

New drugs to treat hepatitis C are expected to be approved over the next few years which promise to cure nearly all patients. However, due to issues of expected drug resistance, suboptimal activity against diverse hepatitis C virus (HCV) genotypes and especially because of their extremely high cost, it is unlikely that these HCV drugs will substantially reduce the world's HCV carrier population of around 170 million in the near future or the estimated global incidence of millions of new HCV infections. For these reasons, there is an urgent need to develop a prophylactic HCV vaccine and also to determine if therapeutic vaccines can aid in the treatment of chronically infected patients. After much early pessimism on the prospects for an effective prophylactic HCV vaccine, our recent knowledge of immune correlates of protection combined with the demonstrated immunogenicity and protective animal efficacies of various HCV vaccine candidates now allows for realistic optimism. This review summarizes the current rationale and status of clinical and experimental HCV vaccine candidates based on the elicitation of cross-neutralizing antibodies and broad cellular immune responses to this highly diverse virus.

Enhanced activation of memory, but not naïve, 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.

Dendritic cells matured by a prostaglandin E2-containing cocktail can produce high levels of IL-12p70 and are more mature and Th1-biased than dendritic cells treated with TNF-α or LPS.

Dendritic cells (DCs) play a crucial role in the initiation of an immune response. As maturation is critical for effective antigen presentation, different methods have been used to generate mature DCs (mDCs) ex vivo. The use of a maturation cocktail (MC) consisting of IL-1ß, IL-6, TNF-α, and prostaglandin E2 (PGE2) initially showed promising results, but then was challenged because of low production of IL-12p70 and the potential for induction of Th2-type immune responses. To investigate this contention, we compared two of the most commonly used maturation factors, TNF-α and LPS, with MC. Maturation cocktail was superior to TNF-α and LPS with respect to enhancement of mDC-specific surface marker expression (CD83, CD86, and HLA-DR), induction of T cell proliferation by mDCs, and directional motility of mDCs toward CCL19. These results were supported by increased expression of a significant number of additional maturation-related genes by MC in comparison to TNF-α and LPS. In addition, we did not observe a Th2-biased shift in the gene expression profile of mDCs generated by MC. Conversely, MC induced more Th1-promoting transcriptional changes than LPS or TNF-α, including increased transcript levels of Th1-type cytokines such as IL-15, IL-12ß, and EBI3 (IL-27ß) and MHC class I molecules, Th1-promoting changes in the transcripts of CXCL16, CCL13, and CCL18, and reduced transcript levels of MHC class II molecules. More interestingly, the Th1-promoting characteristics of MC-mDCs were confirmed by their potential to produce large amounts of IL-12p70 after effective stimulation simulating in vivo events.