Adipose-Derived Stem Cells as Carrier of Pro-Apoptotic Oncolytic Myxoma Virus: To Cross the Blood-Brain Barrier and Treat Murine Glioma.

Treatment of glioblastoma is ineffective. Myx-M011L-KO/EGFP, a myxoma virus actively inducing apoptosis in BTICs linked to recurrence, offers innovative treatment. We loaded this construct into adipose-derived stem cells (ADSCs) to mitigate antiviral host responses and enable systemic delivery. The apoptotic and cytotoxic effects of the construct were studied using murine and human glioblastoma cell lines. Before implementing systemic delivery, we delivered the construct locally using ADSC to verify elimination of orthotopic murine glioma lesions. vMyx-M011L-KO/EGFP was cytotoxic to a murine cell line, preventing effective virus multiplication. In three human glioma cell lines, viral replication did occur, coupled with cell killing. The knock-out construct induced apoptotic cell death in these cultures. ADSCs infected ex vivo were shown to be sufficiently migratory to assure transfer of the therapeutic cargo to murine glioma lesions. Virus-loaded ADSCs applied to the artificial blood-brain barrier (BBB) yielded viral infection of glioma cells grown distally in the wells. Two rounds of local administration of this therapeutic platform starting 6 days post tumor implantation slowed down growth of orthotopic lesions and improved survival (total recovery < 20%). ADSCs infected ex vivo with vMyx-M011L-KO/EGFP show promise as a therapeutic tool in systemic elimination of glioma lesions.

COVID-19 Autopsies: A Case Series from Poland.

This paper presents autopsy findings of 3 COVID-19 patients randomly selected for post-mortem from two tertiary referral Polish hospitals. Analysis of macroscopic, histopathological findings with clinical features was performed. All 3 deceased patients were Caucasian males (average age 61 years, range from 56 to 68 years). Using real-time polymerase chain reaction assay, the patients were confirmed (antemortem) to have severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Two patients were obese, and 1 patient had type 2 diabetes mellitus. The medical history of 1 patient included hemorrhagic pancreatitis, gangrenous cholecystitis, Acinetobacter baumanii sepsis, and cholecystectomy. Pulmonary embolism was diagnosed in 2 patients. At autopsy, in 1 case, the lungs showed bilateral interstitial pneumonia with diffuse alveolar damage (DAD), while in another case, interstitial pulmonary lymphoid infiltrates and enlarged atypical pneumocytes were present but without DAD. Microthrombi in lung vessels and capillaries were observed in 2 cases. This study revealed thrombotic complications of COVID-19 and interstitial pneumonia with DAD presence as the main autopsy findings in patients with SARS-CoV-2 infection that was confirmed antemortem with molecular tests. Autopsy studies using tissue sections handled in accordance with SARS-CoV-2 biosafety guidelines are urgently needed, especially in the case of subjects who were below the age of 60.

Derivatives of Graphene Oxide as Potential Drug Carriers.

Chemically functionalized graphene oxides could be used as novel drug carriers. Covalent alterations of graphene oxides lead to surface changes via formation of chemical bonding while non-covalent ones involve van der Waals forces, hydrogen bonding, and π-π stacking interactions. Covalent modifications appear to be superior as they can yield compounds with desired properties and carriers prepared by other methods are less stable. Synthesis of graphene oxide-iminodiacetic acid and graphene oxide-glycine involves nucleophilic substitution of graphene oxide nanoparticles with iminodiacetic acid or glycine. As the first step, iminodiacetic acid or glycine were transformed into iminodiacetic acid or glycine methyl ester hydrochlorides, respectively, for C-terminus protection. The obtained product, activated in situ, was then used to form amide bonds between graphene oxide and iminodiacetic acid or glycine.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs.IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the nonessential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.

Cyprinid herpesvirus 3: an interesting virus for applied and fundamental research.

Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae is the causative agent of a lethal, highly contagious and notifiable disease in common and koi carp. The economic importance of common and koi carp industries together with the rapid spread of CyHV-3 worldwide, explain why this virus became soon after its isolation in the 1990s a subject of applied research. In addition to its economic importance, an increasing number of fundamental studies demonstrated that CyHV-3 is an original and interesting subject for fundamental research. In this review, we summarized recent advances in CyHV-3 research with a special interest for studies related to host-virus interactions.

Combination of LIGHT (TNFSF14)-Armed Myxoma Virus Pre-Loaded into ADSCs and Gemcitabine in the Treatment of Experimental Orthotopic Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-ß ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.

Removal of Heavy Metal Ions from Wastewaters: An Application of Sodium Trithiocarbonate and Wastewater Toxicity Assessment.

The synthesis and application of sodium trithiocarbonate (Na2CS3) for the treatment of real galvanic wastewater in order to remove heavy metals (Cu, Cd and Zn) was investigated. A Central Composite Design/Response Surface Methodology (CCD/RSM) was employed to optimize the removal of heavy metals from industrial wastewater. Adequacy of approximated data was verified using Analysis of Variance (ANOVA). The calculated coefficients of determination (R2 and R2adj) were 0.9119 and 0.8532, respectively. Application of Na2CS3 conjugated with CCD/RSM allowed Cu, Cd and Zn levels to be decreased and, as a consequence, ∑Cu,Cd,Zn decreased by 99.80%, 97.78%, 99.78%, and 99.69%, respectively, by using Na2CS3 at 533 mg/L and pH 9.7, within 23 min. Implementation of conventional metal precipitation reagents (NaOH, Ca(OH)2 and CaO) at pH 11 within 23 min only decreased ∑Cu,Cd,Zn by 90.84%, 93.97% and 93.71%, respectively. Rotifer Brachionus plicatilis was used to conduct the assessment of wastewater toxicity. Following the application of Na2CS3, after 60 min the mortality of B. plicatilis was reduced from 90% to 25%. Engagement of Na2CS3 under optimal conditions caused the precipitation of heavy metals from the polluted wastewater and significantly decreased wastewater toxicity. In summary, Na2CS3 can be used as an effective heavy metal precipitating agent, especially for Cu, Cd and Zn.

Myxoma Virus Expressing LIGHT (TNFSF14) Pre-Loaded into Adipose-Derived Mesenchymal Stem Cells Is Effective Treatment for Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a weakly immunogenic fatal neoplasm. Oncolytic viruses with dual anti-cancer properties-oncolytic and immune response-boosting effects-have great potential for PDAC management. Adipose-derived stem cells (ADSCs) of mesenchymal origin were infected ex vivo with recombinant myxoma virus (MYXV), which encodes murine LIGHT, also called tumor necrosis factor ligand superfamily member 14 (TNFSF14). The viability and proliferation of ADSCs were not remarkably decreased (1-2 days) following MYXV infection, in sharp contrast to cells of pancreatic carcinoma lines studied, which were rapidly killed by the infection. Comparison of the intraperitoneal (IP) vs. the intravenous (IV) route of ADSC/MYXV administration revealed more pancreas-targeted distribution of the virus when ADSCs were delivered IP to mice bearing orthotopically injected PDAC. The biodistribution, tumor burden reduction and anti-tumor adaptive immune response were examined. Bioluminescence data, used to assess the presence of the luciferase-tagged virus after IP injection, indicated enhanced trafficking into the pancreata of mice bearing orthotopically-induced PDAC, as compared to tumor-free animals, resulting in extended survival of the treated PDAC-seeded animals and in the boosted expression of key adaptive immune response markers. We conclude that ADSCs pre-loaded with transgene-armed MYXV and administered IP allow for the effective ferrying of the oncolytic virus to sites of PDAC and mediate improved tumor regression.

Mesenchymal stem cells as carriers for systemic delivery of oncolytic viruses.

Progress in genetic engineering led to the emergence of some viruses as potent anticancer therapeutics. These oncolytic viruses combine self-amplification with dual antitumor action: oncolytic (destruction of cancer cells) and immunostimulatory (eliciting acquired antitumor response against cancer epitopes). As any other viruses, they trigger antiviral response upon systemic administration. Mesenchymal stem cells are immature cells capable of self-renewing and differentiating into many cell types that belong to three germinal layers. Due to their inherent tumor tropism mesenchymal stem cells loaded with oncolytic virus can improve delivery of the therapeutic cargo to cancer sites. Shielding of oncolytic viral construct from antiviral host immune response makes these cells prospective delivery vehicles to even hard-to-reach metastatic neoplastic foci. Use of mesenchymal stem cells has been criticized by some investigators as limiting proliferative abilities of primary cells and increasing the risk of malignant transformation, as well as attenuating therapeutic responses. However, majority of preclinical studies indicate safety and efficacy of mesenchymal stem cells used as carriers of oncolytic viruses. In view of contradictory postulates, the debate continues. The review discusses mesenchymal stem cells as carriers for delivery of genetically engineered oncolytic constructs and focuses on systemic approach to oncoviral treatment of some deadly neoplasms.

Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma.

Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects.

Combination of vasostatin and cyclophosphamide in the therapy of murine melanoma tumors.

Growth of tumors is strongly dependent upon supply of nutrients and oxygen by de novo formed blood vessels. Inhibiting angiogenesis suppresses growth of primary tumors as well and affects development of metastases. We demonstrate that recombinant MBP/vasostatin fusion protein inhibits proliferation of endothelial cells in vitro. The therapeutic usefulness of such intratumorally delivered recombinant protein was then assessed by investigating its ability to inhibit growth of experimental murine melanomas. In the model of B16-F10 melanoma the MBP/vasostatin construct significantly delayed tumor growth and prolonged survival of treated mice. A combination therapy involving MBP/vasostatin construct and cyclophosphamide was even more effective and led to further inhibition of the tumor growth and extended survival. We show that such combination might be useful in the clinical setting, especially to treat tumors which have already formed microvessel networks.

Conserved Fever Pathways across Vertebrates: A Herpesvirus Expressed Decoy TNF-α Receptor Delays Behavioral Fever in Fish.

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24°C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24°C-32°C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-α, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-α neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness.

Combination of vasostatin gene therapy with cyclophosphamide inhibits growth of B16(F10) melanoma tumours.

Angiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors has important therapeutic implications for the development of novel cancer treatments. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumour growth. In our study, using B16(F10) murine melanoma model and electroporation we attempted intramuscular transfer of human vasostatin gene. The gene therapy was combined with antiangiogenic drug dosing schedule of a known chemotherapeutic (cyclophosphamide). The combination of vasostatin gene therapy and cyclophosphamide administration improved therapeutic effects in melanoma tumours. We observed both significant inhibition of tumour growth and extended survival of treated mice. To our knowledge, this is one of the first reports showing antitumour efficacy of electroporation-mediated vasostatin gene therapy combined with antiangiogenic chemotherapy.

Gene expression profile of B 16(F10) murine melanoma cells exposed to hypoxic conditions in vitro.

Hypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences in hypoxia response between various cell lines. Investigating this phenomenon in melanoma cells contributes to a better understanding of cell lineage-specific hypoxia response and could point out novel hypoxia-regulated genes. We investigated transcriptional activity of B 16(F10) murine melanoma cells cultured for 24 h under hypoxic (nominal 1% O2, 15 samples including controls) and hypoxia-mimicking conditions (cobalt chloride, 100 or 200 microM, 6 samples including controls). Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR <5%) for 1% oxygen experiment and 364 probesets (FDR <5%) for cobalt chloride, which showed differences in expression levels. Analysis of hypoxia-regulated genes (true hypoxia, 1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p < 0.05). The most upregulated genes were Lgals3, Selenbpl, Nppb (more than ten-fold increase). We observed significant differences in expression levels of genes regulating glycolysis (Pfkp, Hk2, Aldo3, Eno2), apoptosis (Bnip3, Bnip31, Cdknla), transcription (Bhlhb2, Sap30, Atf3, Mxil), angiogenesis (Vegfa, Adm, Anxa2, Ctgf), adhesion (Pkp2, Itga4, Mcam), migration (Cnn2, Tmsb4x), and other processes. Both true hypoxia and hypoxia mimicry induced HIF-1-regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences in gene expression between these two experimental conditions. Contrary to hypoxia, cobalt chloride caused suppression of gene expression rather than stimulation, especially concerning transcripts related to proliferation, immune response, DNA repair, and melanin biosynthesis.

Cyprinid Herpesvirus 3: An Archetype of Fish Alloherpesviruses.

The order Herpesvirales encompasses viruses that share structural, genetic, and biological properties. However, members of this order infect hosts ranging from molluscs to humans. It is currently divided into three phylogenetically related families. The Alloherpesviridae family contains viruses infecting fish and amphibians. There are 12 alloherpesviruses described to date, 10 of which infect fish. Over the last decade, cyprinid herpesvirus 3 (CyHV-3) infecting common and koi carp has emerged as the archetype of fish alloherpesviruses. Since its first description in the late 1990s, this virus has induced important economic losses in common and koi carp worldwide. It has also had negative environmental implications by affecting wild carp populations. These negative impacts and the importance of the host species have stimulated studies aimed at developing diagnostic and prophylactic tools. Unexpectedly, the data generated by these applied studies have stimulated interest in CyHV-3 as a model for fundamental research. This review intends to provide a complete overview of the knowledge currently available on CyHV-3.

Antitumour activity of Salmonella typhimurium VNP20047 in B16(F10) murine melanoma model.

A tumour therapy is proposed based on attenuated Salmonella typhimurium VNP20047 expressing the Escherichia coli cytosine deaminase gene. VNP20047 was administered intravenously to B16(F10) melanoma-bearing C57BL/6 mice. VNP20047 proliferated within tumours and livers regardless of the initial inoculum dose. After 10 days the number of bacteria increased in livers up to 4.2 x 10(6) cfu/g and decreased in tumours down to 5.9 x 10(6) cfu/g. VNP20047 at 1 x 10(5) cfu/mouse, when combined with 5-fluorocytosine, inhibited tumour growth by 85% without prolonging animal survival. Histology studies revealed severe lesions in tumours and livers. These data suggest that S. typhimurium VNP20047 induced inflammatory responses, even though the strain was attenuated.

In vivo gene transfer using cetylated polyethylenimine.

UNLABELLED: This report describes gene transfer in vitro as well as in vivo using cetylated low-molecular mass (600 Da) polyethylenimine (28% of amine groups substituted with cetyl moieties), termed CT-PEI. This compound is hydrophobic and has to be incorporated into liposomes in order to be suitable for gene transfer studies. Serum-induced plasmid DNA degradation assay demonstrated that CT-PEI-containing liposomal carriers could protect complexed DNA (probably via condensation). In vitro luciferase gene expression achieved using medium supplemented with 10% serum was comparable to that achieved in serum-reduced medium and was highest for CT-PEI/cholesterol liposomes, followed by CT-PEI/dioleoylphosphatidylcholine liposomes and PEI 600 Da (uncetylated) carrier. In vivo systemic transfer into mice was most efficient when liposome formulations contained CT-PEI and cholesterol. Higher luciferase expression was then observed in lungs than in liver. IN CONCLUSION: liposomes containing cetylated polyethylenimine and cholesterol are a suitable vehicle for investigating systemic plasmid DNA transfer into lungs.

Vasostatin increases oxygenation of B16-F10 melanoma tumors and raises therapeutic efficacy of cyclophosphamide.

One of the preconditions of effective anticancer therapy is efficient transfer of the therapeutic agent (chemotherapeutic) to tumor cells. Fundamental barriers making drug delivery and action difficult include underoxygenation, elevated interstitial pressure, poor and abnormal tumor blood vascular network and acidic tumor milieu. In this study we aimed at developing an optimized scheme of administering a combination of an angiogenesis-inhibiting drug (vasostatin) and a chemotherapeutic (cyclophosphamide) in the therapeutic treatment of mice bearing experimental B16-F10 melanoma tumors. We report that the strongest tumor growth inhibition was observed in mice that received two, three or four vasostatin doses in combination with one injection of cyclophosphamide (i.e., V2 + CTX, V3 + CTX or V4 + CTX schemes). Double administration of vasostatin increases oxygenation of B16-F10 tumors. On the other hand, its five-fold administration lowers tumor oxygenation, breaks down tumor vascular network (increasing hypoxia) and leads in consequence to death of cancer cells and appearance of necrotic areas in the tumor. A decreased cyclophosphamide dose in combination with two doses of vasostatin (V2 + CTX scheme) inhibits tumor growth similarly to a larger dose of cyclophosphamide alone.