The Combinatorial Effect of Ad-IL-24 and Ad-HSV-tk/GCV on Tumor Size, Autophagy, and UPR Mechanisms in Multiple Myeloma Mouse Model.

Multiple myeloma is a type of malignant neoplasia whose treatment has changed over the past decade. This study aimed to investigate the effects of combination of Adenovector-carrying interleukin-24 and herpes simplex virus 1 thymidine kinase/ganciclovir on tumor growth, autophagy, and unfolded protein response mechanisms in mouse model of multiple myeloma. Six groups of mice, including Ad-HSV-tk/GCV, Ad-IL-24, Ad-HSV-tk/IL-24, Ad-GFP, and positive and negative controls, were investigated, and each group was injected every 72 h. The tumor size was measured several times. The expression of LC3B evaluated through western blotting and ASK-1, CHOP, Caspase-3, and ATF-6 genes in the UPR and apoptosis pathways were also analyzed by the quantitative polymerase chain reaction (qPCR) method. The present results showed that the injection of Ad-HSV-tk/GCV, Ad-HSV-tk/IL-24, and metformin reduced the tumor size. The expression of LC3B was significantly higher in the treatment groups and positive control groups compared to the negative control group. The expression of CHOP, caspase-3, and ATF-6 genes was significantly higher in the Ad-IL-24 group compared to the other treatment groups. Besides, the ASK-1 expression was significantly lower in the Ad-IL-24 group as compared to the other groups. Overall, the results indicated that the presence of the HSV-tk gene in the adenovectors reduced the size of tumors and induced autophagy by triggering the expression of LC3B protein. The presence of the IL-24 might affect tumor growth but not as much the therapeutic effect of HSV-tk. Furthermore, the results indicated that co-administration of IL-24 and HSV-tk had no synergistic effect on tumor size control.

Characteristics and outcomes of COVID-19 patients during the BA.5 omicron wave in Tehran, Iran: a prospective observational study.

BACKGROUND: Omicron (B.1.1.529) is the fifth variant of concern of SARS-CoV-2, which has several subvariants. Clinical features of BA.1 and BA.2 infections have been described in the literature, but we have limited information about the clinical profile of BA.5, which caused the seventh wave in Iran. METHODS: A prospective observational study was conducted on the BA.5 confirmed patients referred to Imam Khomeini Hospital Complex, Tehran, Iran, from 11th to 31st August 2022. The patients were divided into the two groups of outpatients and hospitalized patients, and their clinical, radiological, and laboratory data and outcomes were recorded and analyzed. RESULTS: We included 193 patients with confirmed BA.5 infection, of whom 48 patients (24·8%) were hospitalized. The mean age of the patients was 45·3 ± 16·5 years, and 113 patients (58·5%) were female. The mean number of days patients had symptoms was 6·8 ± 2·4 days. The most common symptoms were weakness (69·9%), sore throat (67·4%), myalgia (66·3%), hoarseness (63·7%), headache (55·4%), fatigue (54·9%), and dry cough (50·3%). Fever and dyspnea were significantly more observed in the hospitalized patients (p < 0·0001). The COVID-19 vaccination rate was significantly lower in hospitalized patients than in outpatients (35/48-72·9% vs. 140/145 - 96·6%, p < 0·0001). The most common underlying diseases were hypertension (16·1%), diabetes mellitus (9·8%), and cardiovascular diseases (9·8%), all of which were significantly more common in hospitalized patients. Lung opacities were observed in 81·2% of hospitalized patients. By the end of our study, 1·5% of patients died despite receiving critical care services. CONCLUSIONS: Our findings suggested that BA.5 symptoms are more non-respiratory and usually improve within 7 days. Although the proportion of hospitalized patients is still significant, very few patients require intensive care. COVID-19 vaccination is effective in reducing the hospitalization rate. TRIAL REGISTRATION: Not applicable. This study is not a clinical trial.

Safety and potency of BIV1-CovIran inactivated vaccine candidate for SARS-CoV-2: A preclinical study.

The development of effective and safe COVID-19 vaccines is a major move forward in our global effort to control the SARS-CoV-2 pandemic. The aims of this study were (1) to develop an inactivated whole-virus SARS-CoV-2 candidate vaccine named BIV1-CovIran and (2) to determine the safety and potency of BIV1-CovIran inactivated vaccine candidate against SARS-CoV-2. Infectious virus was isolated from nasopharyngeal swab specimen and propagated in Vero cells with clear cytopathic effects in a biosafety level-3 facility using the World Health Organization's laboratory biosafety guidance related to COVID-19. After characterisation of viral seed stocks, the virus working seed was scaled-up in Vero cells. After chemical inactivation and purification, it was formulated with alum adjuvant. Finally, different animal species were used to determine the toxicity and immunogenicity of the vaccine candidate. The study showed the safety profile in studied animals including guinea pig, rabbit, mice and monkeys. Immunisation at two different doses (3 or 5 µg per dose) elicited a high level of SARS-CoV-2 specific and neutralising antibodies in mice, rabbits and nonhuman primates. Rhesus macaques were immunised with the two-dose schedule of 5 or 3 µg of the BIV1-CovIran vaccine and showed highly efficient protection against 104 TCID50 of SARS-CoV-2 intratracheal challenge compared with the control group. These results highlight the BIV1-CovIran vaccine as a potential candidate to induce a strong and potent immune response that may be a promising and feasible vaccine to protect against SARS-CoV-2 infection.

Diversity, phylogeny and intraspecific variability of Paradiplozoon species (Monogenea: Diplozoidae) parasitizing endemic cyprinoids in the Middle East.

Diplozoidae are common monogenean ectoparasites of cyprinoid fish, with the genus Paradiplozoon being the most diversified. Despite recent studies on Diplozoidae from Europe, Africa and Asia, the diversity, distribution and phylogeny of this parasite group appears to be still underestimated in the Middle East. The objective of this study was to investigate the diversity, endemism and host specificity of diplozoids parasitizing cyprinoid fish from the Middle East, considering this region as an important historical interchange of fish fauna, and to elucidate the phylogenetic position of Middle Eastern Paradiplozoon species within Diplozoidae. Four Paradiplozoon species were collected from 48 out of 94 investigated cyprinoid species. Three known species, Paradiplozoon homoion, Paradiplozoon bliccae and Paradiplozoon bingolensis, were recorded on new cyprinoid host species, and a new species, Paradiplozoon koubkovae n. sp., was recorded on Luciobarbus capito and Capoeta capoeta from the Caspian Sea basin in Iran and Turkey. Paradiplozoon bliccae, exhibiting a wide host range in the Middle East, expressed both morphological and genetic intraspecific variabilities. The four Paradiplozoon species collected in the Middle East were placed in divergent clades, showing the rich evolutionary history of diplozoid parasites in the Middle East. Our study also revealed that two lineages of African diplozoids have a Middle Eastern origin. We stress the importance of applying an integrative approach combining morphological, ecological and molecular methods to reveal the real diversity of diplozoids.

An integrated simulation-optimization framework for assessing environmental flows in rivers.

The present study proposes an integrated simulation-optimization framework to assess environmental flow by mitigating environmental impacts on the surface and ground water resources. The model satisfies water demand using surface water resources (rivers) and ground water resources (wells). The outputs of the ecological simulation blocks of river ecosystem and the ground water level simulation were utilized in a multiobjective optimization model in which six objectives were considered in the optimization model including (1) minimizing losses of water supply (2) minimizing physical fish habitat losses simulated by fuzzy approach (3) minimizing spawning habitat losses (4) minimizing ground water level deterioration simulated by adaptive neuro fuzzy inference system(ANFIS) (5) maximizing macroinvertebrates population simulated by ANFIS (6) minimizing physical macrophytes habitat losses. Based on the results in the case study, ANFIS-based model is robust for simulating key factors such as water quality and macroinvertebrate's population. The results demonstrate the reliability and robustness of the proposed method to balance environmental requirements and water supply. The optimization model increased the percentage of environmental flow in the drought years considerably. It supplies 69% of water demand in normal years, while the environmental impacts on the river ecosystem are minimized. The proposed model balances the portion of using surface water and ground water in water supply considering environmental impacts on both sources. Using the proposed method is recommendable for optimal environmental management of surface water and ground water in river basin scale.

Cell type-specific response of colon cancer tumor cell lines to oncolytic HSV-1 virotherapy in hypoxia.

BACKGROUND: Novel strategies are required since the hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. High mobility group box 1 (HMGB1) protein can block aerobic respiration in cancer cells. We hypothesized that HMGB1could also kill the colorectal cancer cells during hypoxia. METHODS: In this study, we developed oncolytic herpes simplex virus type 1 expressing HMGB1 protein (HSV-HMGB1) and investigated the cytotoxic effect of HSV-HMGB1 and its parental virus (HSV-ble) on three colorectal cancer cells (HCT116, SW480, and HT29) under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. We further identified potential autophagy- related genes in HT29 cells by retrieving mRNA expression microarray datasets from the Gene Expression Omnibus database. These genes were then detected in HT29 cells infected with HSV-HMGB1 and HSV-ble during normoxia and hypoxia by Real-Time quantitative PCR (qRT-PCR). RESULTS: The cytotoxic effect of HSV-HMGB1 was significantly higher than that of HSV-ble during normoxia; however, during hypoxia, HSV-HMGB1 enhanced the viability of HT29 cells at MOI 0.1. Analyzing the cell death pathway revealed that HSV-HMGB1 induced autophagy in HT29 cells under hypoxic conditions. CONCLUSION: In conclusion, it appears that oncolytic virotherapy is cell context-dependent. Therefore, understanding the cancer cells' characteristics, microenvironment, and cell signaling are essential to improve the therapeutic strategies.

Role of autophagy in nerve cell apoptosis in mice infected with street rabies virus.

Rabies is an important zoonotic disease in Iran. Autophagy is a process that maintains homeostasis and can be used as an innate defense mechanism against viruses. Apoptosis is the process of programmed cell death induced by physiological and pathological conditions. The crosstalk of autophagy and apoptosis plays a key role in rabies virus infection. In the current study, NMRI mice intra-cranially received 3-Methyl Adenine (3-MA), rapamycin, street rabies virus (SRABV) and drugs plus SRABV. SRABV and Map1lc3, Beclin-1, Atg5 gene expression were assayed by real-time PCR. Immunohistochemistry was carried out via LC3 protein staining as an autophagy marker, and apoptotic cell death was measured using a TUNEL assay. Map1lc3, Beclin-1 and Atg5 genes expression was significantly increased in drug-plus-SRBV-treated tissues compared to control at 24 hpi. Map1lc3 and Atg5 gene expression showed a slight change in the drugs-plus-virus group compared with the control at 72 hpi. The presence of LC3 in the tissues of the group treated with rapamycin plus SRBV confirmed induction of autophagy, but it was not present in the tissues treated with 3-MA plus SRBV. Our data revealed that apoptosis was induced only in the groups receiving the SRBV or rapamycin or both at 24 hpi. Apoptosis was observed after 72 hours, when the drugs' effect had disappeared in all but the autophagy inhibitor group. Understanding the interaction of SRABV with autophagy pathway genes and its effect on host cell apoptosis may open a new horizon for human intervention and allow a deeper understanding of rabies infections.

Unravelling the stock structure of the Persian brown trout by otolith and scale shape.

The simultaneous use of the scale and otolith morphometry was assessed as a potential tool for the identification of Persian brown trout Salmo trutta stocks of the Lar Lake and five rivers from Lar Basin, Iran. Fourier coefficients (FC) and circularity, rectangularity, roundness, ellipticity and form factor shape indices (SI) were calculated for otolith and scale. Several SIs were significantly different among sites for both structures. Permutational multivariate analysis of variance revealed significant differences between several pairwise comparisons for otolith and scale (FCs and indices separately). Discriminant analysis showed otolith FCs (cross-classification rates: 25-86%) and SI (20-45%) appear to be a relatively acceptable tool to discriminate between several locations. Comparatively, the scale morphometry showed lower discriminatory power (FC = 3-65%; SI = 15-34%), with the exception of SI for Elarm River (60%), Kamardasht River (56%) and Lar Lake (75%). Cross-classification rates improved up to 100% when discriminate analysis incorporating all variables for otolith and scale was performed. The results showed a potential segregation between some water bodies, suggesting that the otolith and scale morphometry could be a useful tool to delimit S. trutta populations in relatively close freshwater environments.

Autophagy induction plays time-dependent role in viral load of HCV infected Huh7.5 cell line.

Autophagy provides an initial membranous platform for incoming hepatitis C virus (HCV) RNA translation and immune evasion. Once HCV replication is established, this infrastructure will be unnecessary for translation of HCV RNA progeny. So, the autophagy plays key role in the replication and immune pathogenesis of HCV virus. The aim of this study was to study the effect of autophagy induction in Huh7.5 cell on virus titer. The Huh7.5 cell was transfected with recombinant pcDNA-Beclin1. The autophagy induction was evaluated via microtubule associated protein 1 light chain 3 staining as autophagy formation marker using flow cytometry. The HCV (JFH1) was inoculated 12-h post-transfection. Next, to evaluate the viral load, viral RNA was extracted after 24 and 48 h and virus titer was calculated using real-time PCR. The result of the current study shows that the induction of autophagy before virus infection was able to enhance virus yield from 4 × 103 copies/mL to 1 × 104 copies/mL at 24-h post-infection, but reduced viral load after 48 h up to 6 × 103 copies/mL. The study of cross-talk between autophagy and HCV may bring new hope for human intervention and treatment of HCV. Also, it opens new avenue to improve virus cultivation in cell culture and understanding HCV and host cell responses. © 2018 IUBMB Life, 71(1):41-44, 2019.

In vitro effect of branched polyethyleneimine (bPEI) on cells infected with human immunodeficiency virus: enhancement of viral replication.

Polyethyleneimine (PEI) is a chemical compound that used is as a carrier in gene therapy/delivery. Some studies have investigated the microbicidal potential and antiviral activity (prophylactic or therapeutic) of PEI and its derivatives. The aim of this study was to investigate the effect of branched polyethyleneimine (bPEI) on human immunodeficiency virus (HIV) replication. Infected cells were treated with bPEI for 36 hours, and the concentration of the viral protein P24 (as a virus replication marker) was determined in cell culture supernatants. This study indicated that bPEI increased HIV replication and decreased the viability of infected cells through cytotoxicity. The toxicity of bPEI its association with and cell death (apoptosis, autophagy and necrosis) have been reported in several studies. To investigate bPEI-induced cytotoxicity, we examined apoptosis and autophagy in cells treated with bPEI, and a significant increase in HIV viral load, the P24 antigen level, autophagy, and necrosis observed. Thus, treatment with bPEI leads to cytotoxicity and higher HIV virus yield.

Autophagy: The multi-purpose bridge in viral infections and host cells.

Autophagy signaling pathway is involved in cellular homeostasis, developmental processes, cellular stress responses, and immune pathways. The aim of this review is to summarize the relationship between autophagy and viruses. It is not possible to be fully comprehensive, or to provide a complete "overview of all viruses". In this review, we will focus on the interaction of autophagy and viruses and survey how human viruses exploit multiple steps in the autophagy pathway to help viral propagation and escape immune response. We discuss the role that macroautophagy plays in cells infected with hepatitis C virus, hepatitis B virus, rotavirus gastroenteritis, immune cells infected with human immunodeficiency virus, and viral respiratory tract infections both influenza virus and coronavirus.

Evaluation of autophagy induction on HEV 239 vaccine immune response in a mouse model.

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4+ and CD8+ T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.

Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-tat-based vaccine candidate to elicit cellular immunity in BALB/c mice.

OBJECTIVE: To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model. RESULTS: Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05). CONCLUSIONS: Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

A novel chimeric influenza virosome containing Vesicular stomatitis G protein as a more efficient gene delivery system.

OBJECTIVES: To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome. RESULTS: A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome. CONCLUSIONS: Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production.

OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Influence of Brucella abortus lipopolysaccharide as an adjuvant on the immunogenicity of HPV-16 L1VLP vaccine in mice.

Brucella abortus lipopolysaccharide (LPS) has less toxicity and no pyrogenic properties in comparison with other bacterial LPS. It is a toll-like receptor 4 agonist and has been shown to have the potential use as a vaccine adjuvant. In this study, the immunostimulatory properties of LPS from smooth and rough strains of B. abortus (S19 and RB51) as adjuvants were investigated for the human papillomavirus type 16 (HPV16) L1 virus-like particles (L1VLPs) vaccines. C57BL/6 mice were immunized subcutaneously three times either with HPV-16 L1VLPs alone, or in combination with smooth LPS (S-LPS), rough LPS (R-LPS), aluminum hydroxide or a mixture of them as adjuvant. The humoral immunity was evaluated by measuring the specific and total IgG levels, and also the T-cell immune response of mice was evaluated by measuring different cytokines such as IFN-γ, TNF-α, IL-4, IL-10 and IL-17. Results showed that serum anti-HPV16 L1VLP IgG antibody titers was significantly higher in mice immunized with a combination of VLPs and R-LPS or S-LPS compared with other immunized groups. Co-administration of HPV-16 L1VLPs with R-LPS elicited the highest levels of splenocytes cytokines (IFN-γ, IL-4, IL-17 and TNF-α) and also effectively induced improvement of a Th1-type cytokine response characterized with a high ratio of IFN-γ/IL-10. The data indicate that B. abortus LPS particularly RB51-LPS enhances the immune responses to HPV-16 L1VLPs and suggests its potential as an adjuvant for the development of a potent prophylactic HPV vaccine and other candidate vaccines.

Suppressive Effects of Chronic Stress on Influenza Virus Protection after Vaccination with Plasmid DNA-Encoded Nucleoprotein.

BACKGROUND: Influenza is a highly infectious and acute respiratory disease caused by an infection of the host respiratory tract mucosa by the influenza virus. The use of DNA vaccines that express conserved genes such as nucleoprotein (NP) represents a new method of vaccination against influenza. In this study, the effect of chronic stress on the efficiency of this type of vaccine has been evaluated in a mouse model. METHODS: The NP DNA vaccine was administered intradermally 3 times on days 0, 3 and 6 to stressed and nonstressed male BALB/c mice. Two weeks after the last immunization, half of these mice were challenged with A/Puerto Rico/8/34 (PR8) influenza virus and were weighed for 12 days, and their mortality rate was assessed during this period. The cellular immune response of the other half of the mice was evaluated by cytotoxicity assay. RESULTS: The results indicate a significant reduction in the cytotoxic T-lymphocyte response of stressed mice in comparison with unstressed mice. Also, the percentage of weight loss and mortality after the challenge in stressed mice was significantly increased compared to the other group. CONCLUSION: These results indicate that the NP DNA vaccine is not able to induce any effective cytotoxic T-lymphocyte response against influenza virus in stressed mice and cannot induce protective immunity against influenza infection in this group of mice.

Nanosilver based anionic linear globular dendrimer with a special significant antiretroviral activity.

HIV is commonly caused to a very complicated disease which has not any recognized vaccine, so designing and development of novel antiretroviral agents with specific application of nanomedicine is a globally interested research subject worldwide. In the current study, a novel structure of silver complexes with anionic linear globular dendrimer was synthesized, characterized and then assessed against HIV replication pathway in vitro as well. The results showed a very good yield of synthesis (up to 70%) for the nano-complex as well as a very potent significant (P < 0.05) antiretroviral activity with non-severe toxic effects in comparison with the Nevirapine as standard drug in positive control group. According to the present data, silver anionic linear globular dendrimers complex may have a promising future to inhibit replication of HIV viruse in clinical practice.