Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses

HOST EPIGENETIC REGULATOR BMI1 AND VIRAL FIBER PROTEIN FOR EQUILIBRIUM BETWEEN ADENOVIRUS AND HOST

Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our projects aim to identify such factors from Ad as well as host cell side, and to clarify their role in regulating Ad propagation and spread. To identity viral factors, we investigated the kinetics of cell killing and Ad propagation following Ad infection at low multiplicity. Our results showed that prior to the release of Ad progenies, Ad infected cells secrete free fiber molecules in an excess, which mask Ad receptor molecules on non-infected bystander host cells, thus preventing these cells from efficient Ad infection and thereby promoting Ad and host cell co-existence. This is advantageous to Ad propagation and persistency of infection compared to the killing of all host cells with rapid kinetics. However, this is disadvantageous if Ad is used as oncolytic agents for therapeutic purposes. To identity host factors, we investigated the effect of polycomb gene BMI1 on Ad propagation. BMI1 is broadly overexpressed in various cancers. By retroviral vector mediated enforced BMI1 overexpression or siRNA mediated down-regulation of endogenous BMI1 expression, we demonstrated an inverse correlation between the Ad progeny production and the levels of BMI1 expression. This effect was not related to the cell cycle status and the receptor dependent Ad infectivity in host cells; nor to the replication of Ad genome and the production of Ad structural proteins. Instead, BMI1 overexpression impaired the morphogenesis of Ad particles, which could be reversed by TSA mediated inhibition of HDAC activity. Our findings indicate overexpression of BMI1 as a limiting factor in cancer therapy based on oncolytic Ad. So inhibition of BMI1 expression or BMI1-related HDAC activity may improve the functionality of oncolytic Ads in cancer therapy. To explore new approaches in Ad vector mediated dual gene expression in human hematopoietic stem cells (HSC), we generated an Ad vector, Ad5F35- ΔLNGFR-BiDp-GFP, encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter. Our data showed that Ad5F35-ΔLNGFR-BiDp vector is highly active in directing dual gene expression in HSCs and most leukemic cell types tested, but the relative levels of dual gene expression by this vector is strongly cell-type dependent

Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells.

OBJECTIVE: Adenoviral vector mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC), when transient gene expression is preferred. Previous studies have demonstrated that fiber retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we have investigated the potential of bi-directional promoter controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. MATERIAL AND METHODS: We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase (PGK) promoter and minimal core promoter from human cytomegalovirus (mCMV). The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types including candidate HSCs was compared to Ad5F35-DeltaLNGFR-IRES vector encoding PGK promoter controlled bicistronic expression cassette for DeltaLNGFR and GFP. RESULTS AND CONCLUSIONS: Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between re-populating progenitor cells. In NOD/SCID mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter controlled Ad5F35 vector such as Ad5F35-DeltaLNGFR-BiDp can be particularly useful for manipulation of myeloid progenitor cells and potentially also in myeloid lineage leukemic cells

Adenovirus assembly is impaired by BMI1-related histone deacetylase activity.

Polycomb ring finger oncogene BMI1 (B cell-specific Moloney murine leukemia virus integration site 1) plays a critical role in development of several types of cancers. Here, we report an inverse relationship between levels of BMI1 expression and adenovirus (Ad) progeny production. Enforced BMI1 expression in A549 cells impaired Ad progeny production. In contrast, knocking-down of endogenous BMI1 expression enhanced progeny production of a conditionally replicating Ad and wild-type Ad5 and Ad11p. Ad vectors overexpressing BMI1 were not impaired in the replication of progeny genomes and in the expression of E1A and Ad structural proteins. However, 293 cells infected by Ad vector overexpressing BMI1 contained a large proportion of morphologically irregular Ad particles. This effect was reversed in 293 cells pre-treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in parallel with the production of infectious Ad particles. Our findings suggest an inhibitory role of BMI1 in Ad morphogenesis that can be implied in Ad tropism and Ad-mediated cancer therapy

The application of approaches in detecting ferroptosis

Ferroptosis is a regulatory cell death (RCD) caused by iron-dependent lipid peroxidation, which is the backbone of regulating various diseases such as tumor, nervous system diseases and so on. Despite ferroptosis without specific detection methods currently, there are numerous types of detection technology commonly used, including flow cytometry, cell activity assay, microscopic imaging, western blotting, quantitative polymerase chain reaction (qPCR). In addition, ferroptosis could be detected by quantifying oxygen-free radicals reactive oxygen species (ROS), the lipid metabolite (malondialdehyde ((MDA)), related pathways and observing mitochondrial damage. In the face of numerous detection methods, how to choose appropriate detection methods based on experimental purposes has become a problem that needs to be solved at present. In this review, we summarized the commonly used detection methods of the critical substances in the process of ferroptosis, in the hope of facilitating the comprehensive study of ferroptosis, with a view to providing a guidance for subsequent related research

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana. The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants

RAGE Deficiency Impairs Bacterial Clearance in Murine Staphylococcal Sepsis, but Has No Significant Impact on Staphylococcal Septic Arthritis.

Septic arthritis is a serious joint disease often caused by Staphylococcus aureus (S. aureus). Receptor for Advanced Glycation End products (RAGE) has an important role in several infections. We sought to investigate the role of RAGE in staphylococcal septic arthritis and sepsis in mice.Wild-type (WT) and RAGE deficient (RAGE-/-) mice were intra-articularly or intravenously inoculated with an arthritic or septic dose of S. aureus LS-1 strain. Clinical arthritis, weight development and mortality were monitored for 14 days. Serum levels of cytokines, kidney bacterial loads as well as micro-CT and histopathology of the joints were assessed.RAGE-/- mice with septic arthritis had significantly lower IL-17A and higher bone mineral density (BMD) compared to the control group. However, no significant differences between the groups were observed regarding the weight loss, the severity and frequency of arthritis, and bacterial loads in the kidneys. In mice with sepsis, the overall mortality rate was similar in RAGE-/- (39%) and in WT mice (45%). However, RAGE-/- mice with sepsis had significantly higher bacterial load in their kidneys compared to the WT controls. In line with data from hematogenous S. aureus arthritis, RAGE deficiency had no impact on arthritis severity in local joint infection.Our results indicate that lack of RAGE has no significant impact on septic arthritis. However, RAGE-/- mice had significantly higher BMD compared to WT mice, which coincided with lower IL-17A in RAGE-/- mice. In sepsis, RAGE deficiency impairs bacterial kidney clearance

Tissue plasminogen activator coating on surface of implants reduces S. aureus biofilm formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.status: publishe

Concomitant use of Ad5/35 chimeric oncolytic adenovirus with TRAIL gene and taxol produces synergistic cytotoxicity in gastric cancer cells

Chimeric adenoviral vectors possessing fiber derived from human adenovirus subgroup B (Ad35) have been developed for their high infection efficiency in cell types which are refractory to adenovirus serotype 5 (Subgroup C) The present study constructed an E1B-deleted chimeric oncolytic adenovirus, SG235-TRAIL, which carries a human TRAIL gene expression cassette and whose fiber shaft and knob domains are from serotype AM. It was found that SG235-TRAIL preferentially replicated in gastric cancer cell lines, SGC-7901 and BGC-823 compared to in normal human fibroblast BJ cells. Also, when compared with a replication-deficient chimeric vector Ad5/35-TRAIL, SG235-TRAIL mediated a higher level of the transgene expression via viral replication in the cancer cells. Further, because of the more efficient cell-entry and infection, SG235-TRAIL induced stronger cell apoptosis than the Ad5 CRAD vector, ZD55-TRAIL In addition, SG235-TRAIL in combination with the chemotherapeutic drug, taxol, produced a synergistic cytotoxic effect in cancer cells in vitro without causing significant toxicity to normal cells. In the gastric tumor xenograft mouse model, intratumoral SG235-TRAIL injection produced a significant antitumor effect 14 days after treatment. Pathological examination demonstrated TRAIL expression and associated apoptosis in majority of SG235-TRAIL-treated tumor cells. These results suggest that SG235-TRAIL is a potential novel, efficient anti-cancer agent, and in combination with taxol, it would be even more useful with considerably low toxic side effects. (C) 2009 Elsevier Ireland Ltd. All rights reserved

The attenuating effect of CTLA4 Ig and anti-TNF therapies on skin lesions was reproducible in milder <i>S</i>. <i>aureus</i> skin infection without affecting local bacterial load.

<p>NMRI mice inoculated with a low dose of <i>Staphylococcus aureus</i> SH1000 (1.0×10⁶ colony-forming units/site) were treated with abatacept (CTLA4-Ig; 0.25 mg/g of body weight; n = 7), etanercept (anti-TNF; 5 μg/g of body weight; n = 7), or phosphate-buffered saline (PBS; n = 7) twice weekly starting on day 7 before inoculation and continuing until the animals were euthanized on day 4. The skin lesion size (<b>a</b>) was observed for 4 days. The frequency of skin necrosis (<b>b</b>) and the bacterial load in skin (<b>c</b>) were analyzed on day 4 after infection. Statistical evaluations were performed using the Mann–Whitney <i>U</i> test and the Fisher exact test. Data are the mean ± SEM. *<i>P</i> < .05, ns = no significance. PBS, phosphate-buffered saline.</p