Pokeweed antiviral protein down-regulates Wnt/β-catenin signalling to attenuate liver fibrogenesis in vitro and in vivo

ABSTRACT A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102(151AA152) and FLP-105(191AA192) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents.

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Mucosal Toxicity Studies of a Gel Formulation of Native Pokeweed Antiviral Protein

Toxicologic Pathology ◽

10.1080/01926230490274362 ◽

2004 ◽

Vol 32 (2) ◽

pp. 212-221 ◽

Cited By ~ 17

Author(s):

Osmond J. D'Cruz ◽

Barbara Waurzyniak ◽

Fatih M. Uckun

Keyword(s):

Clinical Chemistry ◽

Epithelial Tissue ◽

Pokeweed Antiviral Protein ◽

Antiviral Protein ◽

Vaginal Tissue ◽

Polycarbonate Membrane ◽

Membrane Filters ◽

Histologic Evaluation

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a promising nonspermicidal broad-spectrum antiviral microbicide. This study evaluated the mucosal toxicity potential of native PAP in the in vivo rabbit vaginal irritation model as well as the in vitro reconstituted human vaginal epithelial tissue model. Twenty-two New Zealand white rabbits in 4 subgroups were exposed intravaginally to a gel with and without 0.01, 0.1, or 1.0% native PAP for 10 consecutive days. The dose of PAP used represented nearly 200- to 20,000 times its in vitro anti-HIV IC50 value. Animals were euthanized on day 11 and vaginal tissues were evaluated for histologic and immunohistochemical evidence of mucosal toxicity, cellular inflammation, and hyperplasia. Blood was analyzed for changes in hematology and clinical chemistry profiles. Reconstituted human vaginal epithelial tissue grown on membrane filters was exposed to 0.1, 0.1, or 1.0% native PAP in medium or topically via a gel for 24 hours and tissue damage was evaluated by histological assessment. In the in vivo rabbit vaginal irritation model, half of all PAP-treated rabbits (8/16) exhibited an acceptable range of vaginal mucosal irritation (total score <8 out of a possible 16), whereas nearly a third of PAP-treated rabbits (5/16) developed moderate to marked vaginal mucosal irritation (total score >11). However, no treatment-related adverse effects were seen in hematological or clinical chemistry measurements. Furthermore, in vitro exposure of a 3-dimensional human vaginal tissue grown on polycarbonate membrane filters to identical concentrations of PAP either added to culture medium or applied topically via gel formulation did not result in direct toxicity as determined by histologic evaluation. These findings indicate careful monitoring of vaginal irritation will be required in the clinical development of PAP as a nonspermicidal microbicide.

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In vitro and in vivo antileukemic activity of B43-pokeweed antiviral protein against radiation-resistant human B-cell precursor leukemia cells

Blood ◽

10.1182/blood.v86.11.4228.bloodjournal86114228 ◽

1995 ◽

Vol 86 (11) ◽

pp. 4228-4233 ◽

Cited By ~ 23

Author(s):

KG Waddick ◽

DE Myers ◽

R Gunther ◽

LM Chelstrom ◽

M Chandan-Langlie ◽

...

Keyword(s):

B Cell ◽

Severe Combined Immunodeficiency ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

Pokeweed Antiviral Protein ◽

Antiviral Protein ◽

Cell Precursor ◽

Radiation Resistant

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and represents one of the most radiation-resistant forms of human malignancy. In this study, we examined the antileukemic efficacy of the B43 (anti-CD19)-pokeweed antiviral protein (B43-PAP) immunotoxin against radiation-resistant BCP leukemia cells. B43-PAP caused apoptosis of radiation-resistant primary BCP leukemia cells, killed greater than 99% of radiation-resistant primary leukemic progenitor cells from BCP leukemia patients, and conferred extended survival to severe combined immunodeficiency (SCID) mice xenografted with radiation- resistant human BCP leukemia. Furthermore, the combination of B43-PAP and total body irradiation (TBI) was more effective than TBI alone in two SCID mouse bone marrow transplantation models of radiation- resistant human BCP leukemia. Thus, B43-PAP may prove useful in the treatment of radiation-resistant BCP leukemia.

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Inhibition of Japanese encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein

Virus Research ◽

10.1016/j.virusres.2012.10.032 ◽

2013 ◽

Vol 171 (1) ◽

pp. 89-96 ◽

Cited By ~ 24

Author(s):

Hassan Z.A. Ishag ◽

Chen Li ◽

Li Huang ◽

Ming-xia Sun ◽

Bo Ni ◽

...

Keyword(s):

Virus Infection ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Pokeweed Antiviral Protein ◽

Antiviral Protein ◽

Japanese Encephalitis Virus Infection

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TXU (Anti-CD7)-Pokeweed Antiviral Protein as a Potent Inhibitor of Human Immunodeficiency Virus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.2.383 ◽

1998 ◽

Vol 42 (2) ◽

pp. 383-388 ◽

Cited By ~ 31

Author(s):

Fatih M. Uckun ◽

Lisa M. Chelstrom ◽

Lisa Tuel-Ahlgren ◽

Ilker Dibirdik ◽

James D. Irvin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Scid Mouse ◽

Pokeweed Antiviral Protein ◽

Antiviral Protein ◽

Cynomolgus Monkeys ◽

Immunodeficiency Virus ◽

Scid Mouse Model ◽

Anti Hiv ◽

Hiv 1

ABSTRACT We have evaluated the clinical potential of TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate (TXU-PAP) as a new biotherapeutic anti-human immunodeficiency virus (anti-HIV) agent by evaluating its anti-HIV type 1 (anti-HIV-1) activity in vitro, as well as in a surrogate human peripheral blood lymphocyte-severe combined immunodeficient (Hu-PBL-SCID) mouse model of human AIDS. The present report documents in a side-by-side comparison the superior in vitro anti-HIV-1 activity of TXU-PAP compared to the activities of zidovudine, 2′,3′-didehydro-2′,3′-dideoxythymidine, unconjugated PAP, and B53-PAP, an anti-CD4-PAP immunoconjugate. Notably, TXU-PAP elicited potent anti-HIV activity in the Hu-PBL-SCID mouse model of human AIDS without any side effects and at doses that were very well tolerated by cynomolgus monkeys. Furthermore, plasma samples from TXU-PAP-treated cynomolgus monkeys showed potent anti-HIV-1 activity in vitro.

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Transcriptional factor ATF3 promotes liver fibrosis via activating hepatic stellate cells

Cell Death and Disease ◽

10.1038/s41419-020-03271-6 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Zhemin Shi ◽

Kun Zhang ◽

Ting Chen ◽

Yu Zhang ◽

Xiaoxiao Du ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

In Vitro Study ◽

Stellate Cells ◽

Protective Role ◽

Activating Transcription Factor 3 ◽

Dependent Manner ◽

Liver Fibrogenesis

AbstractThe excessive accumulation of extracellular matrix (ECM) is a key feature of liver fibrosis and the activated hepatic stellate cells (HSCs) are the major producer of ECM proteins. However, the precise mechanisms and target molecules that are involved in liver fibrosis remain unclear. In this study, we reported that activating transcription factor 3 (ATF3) was over-expressed in mice and human fibrotic livers, in activated HSCs and injured hepatocytes (HCs). Both in vivo and in vitro study have revealed that silencing ATF3 reduced the expression of pro-fibrotic genes and inhibited the activation of HSCs, thus alleviating the extent of liver fibrosis, indicating a potential protective role of ATF3 knockdown. However, ATF3 was not involved in either the apoptosis or proliferation of HCs. In addition, our data illustrated that increased nuclear localization of ATF3 promoted the transcription of fibrogenic genes and lnc-SCARNA10, which functioned as a novel positive regulator of TGF-β signaling in liver fibrogenesis by recruiting SMAD3 to the promoter of these genes. Interestingly, further study also demonstrated that lnc-SCARNA10 promoted the expression of ATF3 in a TGF-β/SMAD3-dependent manner, revealing a TGF-β/ATF3/lnc-SCARNA10 axis that contributed to liver fibrosis by activating HSCs. Taken together, our data provide a molecular mechanism implicating induced ATF3 in liver fibrosis, suggesting that ATF3 may represent a useful target in the development of therapeutic strategies for liver fibrosis.

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BMP-9 Modulates the Hepatic Responses to LPS

Cells ◽

10.3390/cells9030617 ◽

2020 ◽

Vol 9 (3) ◽

pp. 617

Author(s):

Haristi Gaitantzi ◽

Julius Karch ◽

Lena Germann ◽

Chen Cai ◽

Vanessa Rausch ◽

...

Keyword(s):

Liver Damage ◽

Human Liver ◽

Liver Sinusoidal Endothelial Cells ◽

Inflammatory Reactions ◽

Morphogenetic Protein ◽

Liver Fibrogenesis ◽

Nfkb Pathway ◽

Pre Treatment

It was previously shown that Bone Morphogenetic Protein (BMP)-9 is constitutively produced and secreted by hepatic stellate cells (HSC). Upon acute liver damage, BMP-9 expression is transiently down-regulated and blocking BMP-9 under conditions of chronic damage ameliorated liver fibrogenesis in C57BL/6 mice. Thereby, BMP-9 acted as a pro-fibrogenic cytokine in the liver but without directly activating isolated HSC in vitro. Lipopolysaccharide (LPS), an endotoxin derived from the membrane of Gram-negative bacteria in the gut, is known to be essential in the pathogenesis of diverse kinds of liver diseases. The aim of the present project was therefore to investigate how high levels of BMP-9 in the context of LPS signalling might result in enhanced liver damage. For this purpose, we stimulated human liver sinusoidal endothelial cells (LSEC) with LPS and incubated primary human liver myofibroblasts (MF) with the conditioned medium of these cells. We found that LPS led to the secretion of factors from LSEC that upregulate BMP-9 expression in MF. At least one of these BMP-9 enhancing factors was defined to be IL-6. High BMP-9 in turn, especially in combination with LPS stimulation, induced the expression of certain capillarization markers in LSEC and enhanced the LPS-mediated induction of pro-inflammatory cytokines in primary human macrophages. In LSEC, pre-treatment with BMP-9 reduced the LPS-mediated activation of the NfkB pathway, whereas in macrophages, LPS partially inhibited the BMP-9/Smad-1 signaling cascade. In vivo, in mice, BMP-9 led to the enhanced presence of F4/80-positive cells in the liver and it modulated the LPS-mediated regulation of inflammatory mediators. In summary, our data point to BMP-9 being a complex and highly dynamic modulator of hepatic responses to LPS: Initial effects of LPS on LSEC led to the upregulation of BMP-9 in MF but sustained high levels of BMP-9 in turn promote pro-inflammatory reactions of macrophages. Thereby, the spatial and timely fine-tuned presence (or absence) of BMP-9 is needed for efficient wound-healing responses in the liver.

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135 Jnk1 IN HEPATIC STELLATE CELLS MODULATES LIVER FIBROGENESIS IN VIVO AND IN VITRO

Journal of Hepatology ◽

10.1016/s0168-8278(13)60137-3 ◽

2013 ◽

Vol 58 ◽

pp. S59-S60

Author(s):

F.J. Cubero ◽

G. Zhao ◽

M. Hatting ◽

Y.A. Nevzorova ◽

F. Schaefer ◽

...

Keyword(s):

Hepatic Stellate Cells ◽

Stellate Cells ◽

Liver Fibrogenesis

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Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

10.1101/322040 ◽

2018 ◽

Author(s):

Yasser Hassan ◽

Sherry Ogg ◽

Hui Ge

Keyword(s):

Protein Synthesis ◽

Expression System ◽

Therapeutic Index ◽

Antiviral Protein ◽

E Coli ◽

Protein Isoform ◽

Antiviral Proteins ◽

A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

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SIRT1 ameliorates premature senescence-induced defenestration in hepatic sinusoidal endothelial cell

10.1101/2020.04.24.059048 ◽

2020 ◽

Author(s):

Xiaoying Luo ◽

Yangqiu Bai ◽

Shuli He ◽

Xiaoke Jiang ◽

Zhiyu Yang ◽

...

Keyword(s):

Liver Fibrosis ◽

Oxidative Damage ◽

Adenovirus Vector ◽

Sirtuin 1 ◽

Premature Senescence ◽

Liver Fibrogenesis ◽

Hepatic Sinusoidal Endothelial Cell ◽

Hepatic Sinusoidal Endothelial Cells

AbstractPremature senescence, linked to progerin, involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the potential mechanisms of premature senescence in defenestration in hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects fenestrae remains elusive. Our study showed that in vivo, premature senescence occurred, with decrease of SIRT1, during CCl4-induced defenestration in HSECs and liver fibrogenesis; whereas overexpressing SIRT1 with adenovirus vector lessened progerin-associated premature senescence to relieve CCl4-induced defenestration and liver fibrosis. In vitro, fenestrae in HSECs disappeared, with progerin-associated premature senescence; these effects aggravated by H2O2-induced oxidative damage. Nevertheless, knockdown of NOX2 or overexpression of SIRT1 with adenovirus vector reduced progerin-associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co-localized with accumulation of actin filament (F-actin) in the nuclear envelope of H2O2-treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1. In conclusion, NOX2-dependent oxidative damage aggravates defenestration in HSECs via progerin-associated premature senescence; SIRT1-mediated deacetylation of p53 maintains fenestrae and attenuates liver fibrogenesis through inhibiting premature senescence.

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