Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Human T-cell lymphotropic virus type 1 (HTLV-1) grip on T-cells: investigating the viral tapestry of activation.

INTRODUCTION: Human T-cell Lymphotropic virus type 1 (HTLV-1) belongs to retroviridae which is connected to two major diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell leukemia/lymphoma (ATLL). This study aims to investigate the mRNA expressions of key proteins correlated to T-cell activation in asymptomatic carriers (ACs) HTLV-1 infected patients, shedding light on early molecular events and T-cell activation following HTLV-1 infection. MATERIAL AND METHODS: The study involved 40 participants, including 20 ACs and 20 healthy subjects. Blood samples were collected, ELISA assessment for screening and confirmation with PCR for Trans-activating transcriptional regulatory protein (Tax) and HTLV-1 basic leucine zipper factor (HBZ) of the HTLV-1 were done. mRNA expressions of C-terminal Src kinase (CSK), Glycogen Synthase Kinase-3 Beta (GSK3ß), Mitogen-Activated Protein Kinase 14 (MAP3K14 or NIK), Phospholipase C Gamma-1 (PLCG1), Protein Tyrosine Phosphatase non-Receptor Type 6 (PTPN6) and Mitogen-Activated Protein Kinase Kinase Kinase-7 (SLP-76) and Mitogen-Activated Protein Kinase14 (MAP3K7 or TAK1) were assayed using RT-qPCR. Statistical analyses were performed using PRISM and SPSS software. RESULTS: While there were no significant upregulation in CSK and PTPN6 in ACs compared to healthy individuals, expression levels of GSK3ß, MAP3K14, PLCG1, SLP-76, and TAK1 were significantly higher in ACs compared to healthy subjects which directly contributes to T-cell activation in the HTLV-1 ACs. CONCLUSION: HTLV-1 infection induces differential mRNA expressions in key proteins associated with T-cell activation. mRNAs related to T-cell activation showed significant upregulation compared to PTPN6 and CSK which contributed to T-cell regulation. Understanding these early molecular events in ACs may provide potential markers for disease progression and identify therapeutic targets for controlling viral replication and mitigating associated diseases. The study contributes novel insights to the limited literature on T-cell activation and HTLV-1 pathogenesis.

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

EBNA1 Upregulates P53-Inhibiting Genes in Burkitt's Lymphoma Cell Line.

Background: Suppression of p53 is an important mechanism in Epstein-Barr virus associate-tumors and described as EBNA1-USP7 which is a key axis in p53 suppression. Thus, in this study, we aimed to evaluate the function of EBNA1 on the expression of p53-inhibiting genes including HDAC-1, MDM2, MDM4, Sirt-3, and PSMD10 and the influence of USP7 inhibition using GNE-6776 on p53 at protein/mRNA level. Methods: The electroporation method was used to transfect the BL28 cell line with EBNA1. Cells with stable EBNA1 expression were selected by Hygromycin B treatment. The expression of seven genes, including PSMD10, HDAC-1, USP7, MDM2, P53, Sirt-3, and MDM4, was evaluated using a real-time PCR assay. For evaluating the effects of USP7 inhibition, the cells were treated with GNE-6776; after 24 hours and 4 days, the cells were collected and again expression of interest genes was evaluated. Results: MDM2 (P=0.028), MDM4 (P=0.028), USP7 (P=0.028), and HDAC1 (P=0.015) all showed significantly higher expression in EBNA1-harboring cells compared to control plasmid transfected cells, while p53 mRNA expression was only marginally downregulated in EBNA1 harboring cells (P=0.685). Four-day after treatment, none of the studied genes was significantly changed. Also, in the first 24-hour after treatment, mRNA expression of p53 was downregulated (P=0.685), but after 4 days it was upregulated (P=0.7) insignificantly. Conclusion: It seems that EBNA1 could strongly upregulate p53-inhibiting genes including HDAC1, MDM2, MDM4, and USP7. Moreover, it appears that the effects of USP7 suppression on p53 at protein/mRNA level depend on the cell nature; however, further research is needed.

A New Insight Into p53-Inhibiting Genes in Epstein­Barr Virus-Associated Gastric Adenocarcinoma

Background: The p53 mutation is uncommon in Epstein­Barr virus-linked gastric carcinoma, but its suppression occurs through mechanisms such as ubiquitin specific peptidase 7 (USP7) inhibitions via Epstein­Barr virus nuclear antigen-1 (EBNA1) activity. This study aimed to evaluate the effect of EBNA1 on p53-inhibiting gene expression and the impact of USP7 inhibition on p53 suppression. Methods: MKN-45 cells were transfected with the EBNA1 plasmid. A stable EBNA1 expression cell line was developed through selection based on hygromycin B resistance. Murine double minute (MDM)4, MDM2, sirtuin (SIRT)3, histone deacetylase (HDAC)1, proteasome 26S subunit, Non-ATPase (PSMD)10, USP7, and p53 expression were checked using real-time PCR. Also, cells containing EBNA1 or control plasmid were treated with GNE-6776, and the expression of the interested genes and cell survival were assessed. Results: MDM4, MDM2, and PSMD10 were significantly upregulated in the MKN-45 cell line following EBNA1 transfection. Morphological changes were observed in the cells harboring EBNA1 after 20 days. In the control cells, USP7 inhibition significantly upregulated the HDAC1, PSMD10, MDM4, and MDM2 genes after 24 h, but downregulated these genes after four days. In the EBNA1-harboring cells, MDM2, MDM4, and PSMD10 genes were significantly upregulated after 24 h, and this effect was sustained for all genes except for MDM4, even after four days. Furthermore, USP7 inhibition induced apoptosis in both cell groups. Conclusion: EBNA1 enhances the expression of p53-inhibiting genes. Two events­p53 protein overexpression and apoptosis activation­followed the suppression of the USP7 protein and provided evidence for its possible function. The significance of the EBNA1-USP7 interaction in p53 suppression warrants additional investigation and possibly reconsideration.

EBV and HPV Infections in Colorectal Cancer and Their Effect on P53 and P16 Protein Expression.

Viral infections contribute to 15-20% of newly diagnosed cancers worldwide. There is evidence of a possible etiological role of Epstein-Barr virus (EBV) and high-risk human papillomaviruses (HR-HPVs) in colorectal carcinoma (CRC). Loss of p53 and p16 function has been found in many cancers and this may occur in many different ways, including gene mutation or interaction with viral oncoproteins. This study aimed to evaluate the presence of EBV and HPV in CRC patients in northern Iran and to assess p53 and p16 protein expression related to these viral infections. Real-time PCR was used to amplify the DNA sequences of these viruses in 55 colorectal tumoral tissues, along with their corresponding non-tumoral adjacent tissues. Additionally, immunohistochemistry (IHC) was utilized to determine p53 and p16 protein expression. EBV DNA was detected in 49.1% of CRC tissues. Furthermore, HPV DNA was present in 7.3% of CRC tissues. Notably, the prevalence of EBV infection in tumoral tissues was significantly higher than in non-tumoral tissues (P=0.001). The EBV DNA polymerase catalytic subunit (BALF5) copy number in tumoral tissues was higher than in non-tumoral tissues and this difference was statistically significant (P=0.008). P53 was positive in 21/26 (80.8%) EBV-positive and in 11/25 (44%) EBV-negative samples and this difference was significant (P=0.007). P16 was positive in 13/26 (50%) EBV-positive and in 14/25 (58.3%) EBV-negative samples (P= 0.668). Our findings suggest that EBV infection can increase the risk of CRC. In addition, EBV seems to stabilize p53 in EBV-positive CRC which needs further research. No significant correlation was detected between EBV infection and p16 expression. Also, we could not find a causal relationship between HPV infection and CRC in the study population.

Quantitative analysis of Merkel cell polyomavirus (MCPyV) genome in non-melanoma skin cancer and normal tumor margins.

Merkel cell polyomavirus (MCPyV) is the cause of approximately 80% of Merkel cell carcinomas (MCC). The common types of non-melanoma skin cancer (NMSC) including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are histologically similar to MCC. In the present study, 58 NMSC formalin-fixed paraffin-embedded tissue (FFPE) samples including 12 SCC, 46 BCC, and 58 FFPE samples of adjacent non-tumoral margins as the control were included. Determination of large tumor antigens (LTAg) copy number was performed by qReal-Time PCR as a viral copy number per cell to elucidate MCPyV carcinogenic role in non-melanoma skin cancer. Out of 58 samples, 36 (62%) cancerous and 22 (37.9%) normal tumor margins were positive for MCPyV LTAg. Median copy numbers of MCPyV LTAg among all NMSC samples and non-tumoral margins were 0.308×10-2 and 0.269×10-3 copies per cell respectively (P=0.001). In addition, although the viral load in the majority of samples was detected to be lower than one copy per cell, in 4 BCC samples, a viral load higher than one LTAg copy per cell was detected. The present study revealed that the detection of higher levels of MCPyV LTAg viral load in some BCC and SCC samples may be correlated with the role of MCPyV in some cases of BCC and SCC skin cancer.

Genetic variation in CYP1A1 and AHRR genes increase the risk of systemic lupus erythematosus and exacerbate disease severity in smoker patients.

BACKGROUND: Genetic variations of aryl hydrocarbon receptor (AHR) pathway genes could influence the imbalanced immune response to xenobiotics. Therefore, we aimed to investigate the polymorphism of AHR pathway genes in systemic lupus erythematosus (SLE) patients in association with smoking. METHODS: Genomic DNA from patients (N = 107) and controls (N = 105) of a population from northeast of Iran was used for genotyping of CYP1A1 T>C (rs4646903) and AHRR C>G (rs2292596) variants. The SLEDAI score and smoking status of the patients were registered. The AHR activity was estimated by CYP1A1 and CYP1B1 gene expression in peripheral blood mononuclear cells (PBMC). RESULTS: The C allele in rs4646903 (odds ratio [OR] = 2.67) and G allele in rs2292596 (OR = 1.79) SNPs were significantly associated with the increased risk of SLE. The AHR pathway was more active in high-risk CYP1A1/AHRR: C/G haplotype. The most severe disease was observed in smoker patients with high-risk haplotype and both smoking (Exp (ß) = 9.5) and high-risk CYP1A1/AHRR (C/G) haplotype (Exp (ß) = 3.7) can significantly increase the likelihood of having severe (SLEDAI ≥ 20) SLE disease activity. CONCLUSION: Our findings indicated the association of xenobiotic-metabolizing genes (CYP1A1, AHRR) polymorphisms with the susceptibility to SLE and disease severity regarding the smoking background, suggesting the interaction of gene and environmental risk factors in SLE pathogenesis.

Virus load and incidence of olfactory, gustatory, respiratory, gastrointestinal disorders in COVID-19 patients: A retrospective cohort study.

OBJECTIVES: This study investigated the relationship between viral load and the incidence of olfactory and gustatory dysfunction (OD and GD), the incidence of respiratory and gastrointestinal symptoms and the recovery of OD and GD in COVID-19 patients. DESIGN: A retrospective cohort study. SETTING AND PARTICIPANTS: This study was conducted on 599 outpatients' cases in Golestan province between February and June 2020. MAIN OUTCOME MEASURES: The incidence, severity (complete or partial) and recovery time of OD and GD and their associations with cycle threshold (CT) values of SARS-CoV-2 polymerase chain reaction were assessed. RESULTS: The mean age of patients was 38.27 ± 13.62 years. The incidence of general symptoms included myalgia 70.1%, headache 51.8%, fever 47.7% and dyspnoea 21.4%. 41.9% of patients had gastrointestinal symptoms, including abdominal pain 26.5%, diarrhoea 25.2%, nausea 20.5% and vomiting 12.9%. 12.2% of patients had comorbidity. The trimester recovery rates of OD and GD were 93.94% and 94.74% respectively. The mean recovery time of OD and GD was 14.56 ± 13.37 and 13.8 ± 3.77 days respectively. The mean CT value in all patients was 27.45 ± 4.55. There were significant associations between the mean of CT value with headache (p = 0.04), GD (p = 0.002) and OD (p = 0.001). CONCLUSIONS: The finding of this study indicates a possible association between viral load with incidence of OD and GD in COVID-19 patient's cases and assures the recovery of OD/GD in these patients.

Human gene polymorphisms and their possible impact on the clinical outcome of SARS-CoV-2 infection.

The SARS-CoV-2 pandemic has become one of the most serious health concerns globally. Although multiple vaccines have recently been approved for the prevention of coronavirus disease 2019 (COVID-19), an effective treatment is still lacking. Our knowledge of the pathogenicity of this virus is still incomplete. Studies have revealed that viral factors such as the viral load, duration of exposure to the virus, and viral mutations are important variables in COVID-19 outcome. Furthermore, host factors, including age, health condition, co-morbidities, and genetic background, might also be involved in clinical manifestations and infection outcome. This review focuses on the importance of variations in the host genetic background and pathogenesis of SARS-CoV-2. We will discuss the significance of polymorphisms in the ACE-2, TMPRSS2, vitamin D receptor, vitamin D binding protein, CD147, glucose-regulated protein 78 kDa, dipeptidyl peptidase-4 (DPP4), neuropilin-1, heme oxygenase, apolipoprotein L1, vitamin K epoxide reductase complex 1 (VKORC1), and immune system genes for the clinical outcome of COVID-19.

Viruria of Human BK Virus and John Cunningham Virus among Renal Transplant Recipients and Healthy Control in Southeast of Caspian Sea.

BACKGROUND: Members of the Polyomaviridae family, BK virus (BKV), and John Cunningham virus (JCV) are linked to polyomavirus-associated nephropathy-associated transplant rejection in immunodeficient patients. OBJECTIVE: The aim of the study was to evaluate the prevalence of BKV and JCV in immunocompetent individuals in the north of Iran. METHODS: Ninety-one urine samples were obtained from renal transplant recipients with a mean age of 39.78 ± 11.19 years. A healthy control group of 65 volunteers with an average age of 40.32 ± 10.7 years also contributed. After DNA extraction, positive cases were detected through PCR. Genotyping was done by alignment and phylogenetic tree construction of the VP1 region against all known JCV and BKV genotypes. RESULTS: The prevalence of BKV and JCV was 15.38 and 19.78%, respectively. JCV was detected in 7.69% of the control group. The prevalence of the BKV between the case and control groups was significant (p < 0.0001). There was no significant association between BKV and JCV and duration of dialysis (p > 0.05). Overall, 62.16% of JCV cases were genotype I. Besides, genotype II was dominant within patients with BKV-positive patients. DISCUSSION: The results obtained here show a relatively lower prevalence of BKV and JCV in immunocompromised renal transplant receivers and healthy control than those reported from other areas in Iran. JCV genotyping was evaluated for the first time in Iran. Genotype I for JCV and genotype II for BKV were dominant genotypes in the north of Iran.

COVID-19 in Pediatrics: Demographic, Clinical, Laboratory, and Radiological Characteristics of Infected Patients With SARS-CoV-2.

The COVID-19 disease usually leads to mild infectious disease in children, but some develop serious complications. Here, we describe the characteristics of children with COVID-19 in northern Iran, the Golestan province. Ninety-one confirmed cases were enrolled in the study, aged 0-18 years. Demographic, clinical, comorbidity, laboratory, and radiological data were compared based on the disease severity (admitted to intensive care unit (ICU) or not) and disease outcome (recovered or deceased). Sixteen (17.5%) cases were hospitalized in ICU, and 8/91 (8.8%) deceased. Fever and cough were the most common clinical symptoms. Among all symptoms notified there were no significant differences between severe and milder cases, or between those who deceased and recovered. Failure to thrive (FTT), malignant disease and neurological disease were significantly more prevalent in severe cases as was frequently reported comorbidities. Laterality, ground-glass opacity, and lung consolidation were the most common findings in chest computed tomography. The data confirms that the COVID-19 disease has various presentations in children, and clinical, laboratory, and radiological findings may help predict the development of severe forms of COVID-19 among children.

Cell Death Mechanisms in Esophageal Squamous Cell Carcinoma Induced by Vesicular Stomatitis Virus Matrix Protein.

OBJECTIVES: Vesicular stomatitis virus (VSV) is under development as an oncolytic virus due to its preferential replication in cancer cells and oncolytic activity, however the viral components responsible have not yet been determined. In this study the effects of VSV wild-type (wt) and M51R-mutant matrix proteins (M51R-mMP) on apoptosis, pyroptosis, necroptosis, and autophagy pathways, in an esophagus cancer cell line (KYSE-30) were investigated. METHODS: The KYSE-30 cells were transfected with pcDNA3.1 plasmids encoding wt or M51R-mMP, and apoptosis, pyroptosis, necroptosis, and autophagy were evaluated 48 and 72 hours after transfection. RESULTS: KYSE-30 cells transfected with VSV wt and M51R-mMPs significantly reduced cell viability to < 50% at 72 hours post-transfection. M51R-MP significantly increased the concentration of caspase-8 and caspase-9 at 48 and 72 hours post-transfection, respectively ( p < 0.05). In contrast, no significant changes were detected following transfection with the VSV wt plasmid. Moreover, VSV wt and M51R-mMP transfected cells did not change the expression of caspase-3. VSV wt and M51R-mMPs did not mMP change caspase-1 expression (a marker of pyroptosis) at 48 and 72 hours post-transfection. However, M51R-mMP and VSV wt transfected cells significantly increased RIP-1 (a marker of necroptosis) expression at 72 hours post-infection ( p < 0.05). Beclin-1, a biomarker of autophagy, was also induced by transfection with VSV wt or M51R-mMPs at 48 hours post-transfection. CONCLUSION: The results in this study indicated that VSV exerts oncolytic activity in KYSE-30 tumor cells through different cell death pathways, suggesting that M51R-mMP may potentially be used to enhance oncolysis.

M51R and Delta-M51 matrix protein of the vesicular stomatitis virus induce apoptosis in colorectal cancer cells.

Colorectal cancer (CRC) is the third most common cancer in both men and women. Oncolytic viral-based therapy methods seem to be promising for CRC treatment. Vesicular stomatitis virus (VSV) is considered as a potent candidate in viral therapy for several tumors. VSV particles with mutated matrix (M) protein are capable of initiating cell death cascades while not being harmful to the immune system. In the current study, the effects of the VSV M-protein was investigated on the apoptosis of the colorectal cancer SW480 cell. Wild-type, M51R, and ΔM51 mutants VSV M-protein genes were cloned into the PCDNA3.1 vector and transfected into the SW480 cells. The results of the MTT assay, Western blotting, and Caspase 3, 8, and 9 measurement, illustrated that both wild and M51R mutant M-proteins can destroy the SW480 colorectal cancer cells. DAPI/TUNEL double-staining reconfirmed the apoptotic effects of the M-protein expression. The ΔM51 mutant M-protein is effective likewise M51R, somehow it can be considered as a safer substitution.

Analysis of poliovirus receptor, CD155 expression in different human colorectal cancer cell lines: Implications for poliovirus virotherapy.

CONTEXT: Poliovirus (PV) receptor (CD155) is expressed on several kinds of cells and exerts diverse functions. Various investigations have confirmed that changes in CD155 expression in cancer cell lines affect metastasis, proliferation, and migration. AIMS: The purpose of the present study was to investigate the CD155 transcript and protein expression in human colon adenocarcinoma cell lines in comparison to normal fetal human colon (FHC) cells. MATERIALS AND METHODS: The CD155 expression level in four human adenocarcinoma cell lines and normal colon cell line were assessed using the SYBR green quantitative real-time polymerase chain reaction (PCR) and flowcytometry. RESULTS: The results of real-time PCR indicated that CD155 was significantly overexpressed in all human adenocarcinoma cell lines (P = 0.000). The highest and the lowest expression level of CD155 messenger RNA was observed in SW480 and HT29 cell lines by 491.14, and 12.04 fold changes, respectively, in comparison with the human normal cell line (FHC). Results of flowcytometry indicate that protein was strongly expressed in cancer cell lines. SW480 cells showed the highest CD155 protein expression level of 98.1%, whereas this protein expression was 1.3% in human normal colon cell line (FHC). Totally, these data indicate that CD155 expression is significantly elevated in cancer cell lines. CONCLUSIONS: The preferential expression of CD155 on cancer cell lines rather than on normal cell line suggests that CD155 could be targeted for future PV virotherapy.

Non-replicating Newcastle Disease Virus as an adjuvant for DNA vaccine enhances antitumor efficacy through the induction of TRAIL and granzyme B expression.

The potential of non-replicating Newcastle Disease Virus (NDV) as an adjuvant for DNA vaccination remains to be elucidated. To assess the therapeutic effects of DNA vaccine (HPV-16 E7 gene) adjuvanted with NDV, female C57/BL6 mice were inoculated with murine TC-1 cells of human papillomavirus (HPV)-related carcinoma, expressing human papillomavirus 16 (HPV-16) E6/E7 antigens, and immunized with DNA vaccine alone or pretreated with NDV. One week after third immunization, Cytotoxic T lymphocytes (CTLs), splenocyte proliferation, cytokine balance (IFN-γ, IL-4 and IL-12 secretions) and intratumoral expression of cytotoxicity related proteins in tumor lysates were investigated. The results showed that treatment with non-replicating NDV prior to DNA vaccine induced tumor-specific cytolytic and splenocyte proliferation responses. The levels of cytokines IL-12, IL-4 and IFN-γ after treating with combined E7-DNA -non-replicating NDV (NDV-DNA Vaccine) were significantly higher than those of control groups. The intratumoral granzyme B and Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)-mediated apoptosis was also significantly increased. Tumor therapeutic experiments showed that the NDV pretreatment could reduce the tumor progression of established E7-expressing TC-tumors. Taken together these data suggest that the significant antitumor responses evidenced during treatment with non-replicating NDV prior to DNA vaccine are due, in part, to strong E7-induced cellular immunity and enhanced expression of cytotoxicity related proteins in the tumor microenvironment. These observations indicated the potential of non-replicating NDV as an adjuvant for enhancing therapeutic DNA vaccines -induced immunity and antitumor responses.

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

Antitumor Immunity Induced by Genetic Immunization with Chitosan Nanoparticle Formulated Adjuvanted for HPV-16 E7 DNA Vaccine.

BACKGROUND: In recent years attention has been paid to develop effective adjuvant systems for DNA vaccines. Co-formulation of a gene delivery vector with an immunostimulator can enhance therapeutic efficiency of DNA vaccine. OBJECTIVE: To investigate the efficacy of chitosan as a nanodelivery system to enhance antitumor effects of human papilloma virus (HPV)-16 DNA vaccine with IL-12 gene for protection against TC-1 tumor using an animal model. METHODS: The mice were challenged by subcutaneous injection of TC-1 cells and immunized intramuscularly with DNA vaccine thrice at seven-day intervals. One week after the last immunization, mice were sacrificed and antitumor effects were assessed through measuring lymphocyte proliferation, cytotoxicity, cytokines production, and tumor regression. RESULTS: We found that co-formulation and co-administration of chitosan nanoparticles and IL-12 with HPV-16 E7 DNA vaccine induced higher antitumor effects compared with chitosan or IL-12 alone. E7-specific lymphocyte proliferation index and CTL activity were found to be significantly higher in combination group in comparison to single vaccination with either chitosan or IL-12. Co-formulation of chitosan and IL-12 resulted in higher IFN-γ and IL-4, and decreased IL-10 production. Furthermore, combined vaccination highly inhibited the tumor progression compared with chitosan or IL-12 alone. CONCLUSION: Chitosan nanoparticle is a promising delivery system for DNA vaccine and IL-12 is an effective genetic adjuvant for the induction of strong antitumor immune response.

Oral poliovirus vaccine-induced programmed cell death involves both intrinsic and extrinsic pathways in human colorectal cancer cells.

PURPOSE: Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. Replication-competent viruses, which are naturally able to infect and lyse tumor cells, seem to be promising in this field. The aim of this study was to evaluate the potential of oral poliovirus vaccine (OPV) on human CRC cells and elucidate the mechanism of apoptosis induction. MATERIALS AND METHODS: Protein and gene expression of poliovirus (PV) receptor (CD155) on four human CRC cell lines including HCT116, SW480, HT-29, and Caco-2 and normal fetal human colon (FHC) cell line as a control were examined by flow cytometry and SYBR Green Real-Time PCR, respectively. Cytotoxicity of OPV on indicated cell lines was tested using MTT assay. The ability of OPV on apoptosis induction for both intrinsic and extrinsic pathways was examined using caspase-8 and caspase-9 colorimetric assay kits. The PV propagation in mentioned cell lines was investigated, and the quantity of viral yields (cells associated and extracellular) was determined using TaqMan PCR. RESULTS: CD155 mRNA and protein were expressed significantly higher in studied CRC cell lines rather than the normal cell line (P=0). OPV induced cell death in a time- and dose-dependent manner in human CRC cells. Apoptosis through both extrinsic and intrinsic pathways was detected in CRC cells with the minimum level found in FHC. PV viral load was significantly correlated with apoptosis via extrinsic (R=0.945, P=0.0001) and intrinsic (R=0.756, P=0.001) pathways. CONCLUSION: This study suggests that OPV has potential for clinical treatment of CRC. However further studies in animal models (tumor xenografts) are needed to be certain that it is qualified enough for treatment of CRC.

Mucosal immune parameters, immune and antioxidant defence related genes expression and growth performance of zebrafish (Danio rerio) fed on Gracilaria gracilis powder.

In the present study zebrafish (Danio rerio) has been used as model organism to establish the effects of dietary supplementation of Gracilaria gracilis powder (GP) on mucosal and innate immune parameters, antioxidant enzymes, and growth. In order to establish these features, zebrafish were fed for eight weeks with experimental diets containing different levels of Red algae, 0.25, 0.5 and 1% of GP; also, a group was fed with control diet. At the end of the experimental period the antioxidant superoxide dismutase and catalase (SOD, CAT) genes expression, interleukin 1 beta (il-1ß), lysozyme (LYZ), tumor necrosis factor alpha (TNF-α) for immune-related genes expression, total immunoglobulin (Ig), total protein, alkaline phosphatase (ALP) activity for innate immune parameters, and growth performance have been established. The GP dietary supplementation showed differences in SOD and CAT expression in zebrafish whole body respect to the control group. Non-signifcant differences were noticed among the different groups in case of TNF-α, LYZ and il-1expression (P > 0.05). The skin mucus total Ig and total protein in the group fed on 1% of GP were significantly higher respect to control group (P < 0.05). 0.25 and 0.5% of GP dietary supplementation significantly enhanced skin mucus ALP activity levels (P < 0.05). No significant differences were recorded for growth performances among groups (P > 0.05). The results obtained in the present study revealed that G. gracilis could be takes in account as fishes diet supplementation for its immune system stimulants effects.