Evaluation of antitumor activity in strains of a new rotavirus group in Reoviridae family on a model of transplantable murine melanoma

The purpose was to study antitumor effects of the group K rotaviruses strains No. 228 and No. 100 in the experiment on a model of transplantable murine melanoma. Material and methods. The study included 65 С57Black/6 mice with transplantable B16/F10 melanoma and two strains of the Reoviridae family members characterized as rotaviruses, not belonging to the known groups, with the working title “group K rotaviruses (RVK)”. Animals received RVK in “vaccination” (before tumor transplantation) and “treatment” (after tumor formation) modes. Live and inactivated strains were used. RVK were administered intramuscularly as 0.3 ml of virus-containing culture fluid with at least 5x109 viral particles per 1 ml, with a total of 4 injections. Life span of mice and morphological characteristics of tumors were evaluated. Results. Injections of both strains increased the survival of tumor-bearing mice by 1.7-1.9 times in 4 of 8 experimental groups, compared to controls. The modes of RVK administration showed some differences: the survival was longer in mice with the “vaccination” mode compared to the “treatment” mode. Morphological changes in tumors were similar after application of both modes and inclued dystrophic changes of tumor cells, formation of extensive necrosis areas, and leukocyte infiltration. Discussion. Live and inactivated RVK had unidirectional effects implying its association with immunomodulatory action rather than with a direct lytic effect on the tumor. Conclusions. Both studied strains of rotaviruses in the group K had antitumor effect in the model of transplantable В16/F10 murine melanoma in the «vaccination» mode.

Lytic Bacteriophages and Phage Cocktails Seem to be a Future Alternative Against Multi-Drug Resistant Bacterial Infections

Lytic bacteriophages have the efficacy to act and eradicate pathogenic bacteria as an attractive tool in the near future. Bacteriophages specifically kill multidrug-resistant bacteria even which have the capacity to form biofilms. The present review mainly focused on the efficacy of bacteriophages and cocktails as therapeutic agents against predominate MDR-bacteria and their biofilms which are isolated from septic wound infections. The body of evidence includes data from studies investigating bacteriophages from sewage samples as novel antibacterial and antibiofilm agents against pathogenic bacteria. The goal of this review is to present an overview on predominant bacteria from septic wound infection, the biofilm-forming capacity of bacteria, lytic effect of bacteriophages and phage cocktails with an emphasis on the application of bacteriophages against septic wound causing bacteria.

Oncolytic Virotherapy in Multiple Myeloma: A Possible Alternative Role of Bovine Viruses.

Multiple Myeloma (MM) is a plasma cell malignancy characterized by the tight dependence to the bone marrow (BM) microenvironment that supports MM cells survival. Despite significant therapeutic progress in the recent years with the introduction of several new drugs, MM remains an incurable disease. Oncolytic virotherapy is an alternative therapeutic technology in the cancer treatment exploiting naturally or genetically engineered viruses able to infect, transduce and consequently kill cancer cells directly or indirectly through the delivery of the microenvironment cells. Several oncolytic viruses have shown promising pre-clinical results for the treatment of MM, in particular Measles virus and Reovirus. However, the use of human viruses such as Measles could be limited by the antiviral immune response of the patients due to vaccination or natural infection. In order to avoid these potential limits of the human viruses, the aim of this study was to investigate the development of bovine viruses as an alternative oncolytic strategy in MM by checking these viruses that showed an anti-tumoral activity in different solid tumors. Thus, we investigated the potential lytic effect on human MM cells of the Bovine Viral Diarrhea Virus (BVDV), known to bind CD46 as reported for human measles virus, and the Oncolytic Bovine Herpesvirus type 4 (BoHV-4). Firstly, we treated several human myeloma cell lines (HMCLs) with BVDV or the heat-inactivated virus for 24, 48 and 72 hours. The infection efficiency was verified by nested multiplex PCR. We showed a significant increase of cell mortality, checked by trypan blue count and flow cytometry analysis, already after 48 hours of infection in JJN3 (mean±SD % of dead cells: BVDV 45±11 % vs inactive virus 16±2.5 %, p=0.013), and in OPM2 (BVDV 43±1.4 % vs inactive virus 28±2.1 %, p= 0.015) but not in U266 (BVDV 25±23 % vs inactive virus 18±12 %, p=NS. However, BVDV pre-treatment for 12 hours and followed by 48 hours bortezomib (bor) treatment (concentration ranging: 5-10nM) significantly restored bor sensitivity in U266 resistant cells (mean±SD % of dead cells: BVDV plus bor 10 nM 69±8 % vs inactive virus + bor 10 nM 36±1 %, p= 0.031). Interestingly, the cytotoxic effect of BVDV treatment in HMCLs was associated by a significantly increase of apoptotic markers evaluated by flow cytometry. Subsequently, we infected BM primary purified CD138+, showing a significant increase of the mortality rate after treatment with BVDV as compared to the inactivated virus. On the contrary, BVDV was not able to infect human BM mesenchymal stromal cells (MSCs) not showing any lytic effect. Thereafter the capacity to induce MM cell lysis by a recombinant BoHV-4 virus, delivering a Red Fluorescent Protein (RFP) expression cassette as reporter gene, was also evaluated. As observed by the percentage of RFP-positive cells, BoHV-4 was unable to infect and consequently to kill several HMCLs tested. Then we used BM MSCs as in vitro model for oncolytic virus delivery in co-culture systems with MM cells. BoHV-4 infected hTERT-MSCs, expressing RFP at 24, 48 and 72 hours. Consistently, hTERT-MSCs viability was progressively reduced at 24 and 48 hours after infection, as compared to controls, (mean±SD % reduction of cell viability: -22±8 %, p=0.0254 and -49±2 %, p=0.0001, respectively), reaching the highest effect at 72 hours (-70±1.5 %, p=0.0003). Thus we evaluated the effect of BoHV-4 in a co-culture system between human MSCs and two stroma-dependent HMCLs as INA-6 and saMMi. In both cases the percentage of dead HMCLs increased in co-culture with BoHV-4 infected hTERT-MSCs, as compared to hTERT-MSCs untreated controls (INA-6: BoHV-4 61±2.1 % vs control 12±2.1 %, p= 0.0018; saMMi: BoHV-4 48±1.9 % vs control 14±1.4 %, p= 0.0027). Overall our data indicate both direct and MSC-mediated oncolytic effects of bovine viruses on MM cell, suggesting their possible use as novel alternative anti-MM virotherapy strategy. Disclosures Giuliani: Celgene: Research Funding; Janssen: Research Funding.

Abstract W P366: Hypothermia Does Not Affect Tissue Plasminogen Activator Activity in Acute Ischemic Stroke Patients as Measured by Thromboelastography

Background: Currently, t-PA is the only FDA approved treatment for acute ischemic stroke (AIS). Supplementing t-PA with therapeutic hypothermia is being evaluated, but cooler temperatures may affect the enzymatic activity of t-PA. Thromboelastography (TEG) determines coagulation status in whole blood, and has detected hypercoagulability in AIS patients compared to healthy controls. Using TEG, we evaluated the effect of variable degrees of hypothermia on clot formation and lysis in AIS patients receiving t-PA. Methods: Between June 2012 -July 2013, venous blood from 18 AIS patients receiving t-PA within 4.5 hours of symptom onset was collected prior to and 10 minutes after t-PA bolus. Blood samples were analyzed by TEG at 30°C, 33°C, and 37°C. The variables of interest were R (initiation of clot formation), K (speed of clot strengthening), Angle α (rate of clot formation), and LY30 (percentage of clot lysis over 30 minutes) (see figure). All statistical analyses were performed using SAS 9.3. Results: Baseline R averaged 6.0, 5.6, and 4.6 minutes at 30°C, 33°C, and 37°C (p=0.02),K averaged 2.5, 2.3, and 1.4 (p=0.01),and Angle averaged 59.1, 62.4, and 69.3(p=0.01), indicating slower clotting at lower temperatures. Post t-PA LY30 averaged 93.9, 93.9, 89.8 (p= 0.61, N=18) indicating no effect on t-PA lytic activity at lower temperatures. Conclusions: Our data suggest that hypothermia progressively slows clot formation in AIS patients but has no effect on the lytic effect of t-PA as measured by TEG.

Edwardsiella tarda Ivy, a Lysozyme Inhibitor That Blocks the Lytic Effect of Lysozyme and Facilitates Host Infection in a Manner That Is Dependent on the Conserved Cysteine Residue

ABSTRACTEdwardsiella tardais a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenicE. tardastrain TX01. IvyEtpossesses the Ivy signature motif CKPHDC in the form of82CQPHNC87and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of theivyEtgene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt(rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared.In vitrostudies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme onE. tarda.In vivoanalysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEtis a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

Characterization of bacteriophages with a lytic effect on various Salmonella serotypes and Escherichia coli O157:H7

Four phages isolated from cattle and poultry feces were analyzed for their ability to lyse Salmonella serotypes and Escherichia coli O157:H7. The phage one-step growth curves, morphology, and genetic characteristics were determined. All phages showed a lytic effect on various Salmonella serotypes and E. coli O157:H7, which lysed at least 70% of the 234 strains tested. The phages had latent periods ranging from 10 to 15 min and generation times of 30 to 45 min, while burst size fluctuated between 154 and 426 PFU/cell. Phages morphology showed isometric and elongated heads and rigid contractile tails, consistent with morphology of the Myoviridae family. Phages’ DNA dendrograms showed a distinctive RFLP when digested by HindIII and EcoRV, and SDS–PAGE profile showed distinctive proteins expression as well. In vitro phage challenge showed a total reduction of E. coli O157:H7, Salmonella Typhimurium and Saintpaul counts at 2 h, whereas for Salmonella Montevideo a reduction and retardation growth, at a multiplicity of infection (MOI) of 100, was observed; however, under a MOI of 10 000, no viable cells were detected after 4 h. The wide host ranges of these phages suggested they could be used for simultaneous biocontrol of some Salmonella serotypes and E. coli O157:H7.

Duddingtonia flagrans, Monacrosporium thaumasium and Pochonia chlamydosporia as possible biological control agents of Oxyuris equi and Austroxyuris finlaysoni

The action of four fungal isolates of the species Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34a) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Oxyuris equi and Austroxyuris finlaysoni was evaluated in two assays (A and B). Eggs of O. equi (Test A) and A. finlaysoni (Test B) were plated on Petri dishes with 2% water-agar with grown fungal isolates and control without fungus. After 5, 10 and 15 days, 100 eggs were collected and classified according to the following parameters: type 1 effect, physiological and biochemical effect without morphological damage to the eggshell; type 2 effect, lytic effect with morphological alteration of the eggshell and embryo; and type 3 effect, lytic effect with morphological alteration of the eggshell and embryo, hyphal penetration and internal egg colonization. Pochonia chlamydosporia isolates VC1 and VC4 showed ovicidal activity for type 1, 2 and 3 effects on eggs of O. equi and eggs of A. finlaysoni. In vitro assays A and B showed that P. chlamydosporia had a negative influence on eggs of O. equi and A. finlaysoni and can be considered as a potential biological control agent of nematodes.