Lessons from COVID-19 Pandemic: A Successful Policy and Practice by Pasteur Institute of Iran.

The present study aims to provide an insight to the comprehensive efforts of Pasteur Institute of Iran (PII) regarding COVID-19 management, research, achievements, and vaccine production, though there are many challenges. The relevant literature review was investigated through national and international database and also reports from the related research departments. Six strategies were taken by PII to manage the pandemic of COVID-19. While this pandemic has been hopefully controlled, SARS-CoV-2 could still be a potential threat. Therefore, COVID-19 data management and updated studies, as well as long-term safety and efficacy of the SARS-CoV-2 vaccines are still on the agenda.

Assessment the Efficiency of the Constructed Minigenome of Rabies Virus using PV Strain as Helper Virus.

INTRODUCTION: Rabies is an acute viral disease that causes encephalomyelitis in mammals and human. The only way to prevent this disease is through vaccination before or after exposure. The aim of this study is to evaluate the efficiency of the Pasteur virus (PV) minigenome, using PV strain. MATERIALS AND METHODS: Enhanced Green Fluorescent Protein (EGFP) sequence was placed between the designed necessary elements (Hammerhead, HDV ribozyme, 3' Leader, and 5' Trailer sequences), which resemble the rabies virus PV strain (PV2061) genome and anti-genome. These constructs were placed between T7 polymerase promoter and T7 polymerase terminator sequences. The accuracy of the minigenome was confirmed by the expression of EGFP using the helper virus in T7-BHK cell line. RESULTS: The viral necessary elements of positive and negative sense strands were evaluated for the ability of EGFP expression in the presence of the helper virus. While the positive strand showed background results, no EGFP background was observed in the negative strand application. CONCLUSION: Establishment of minigenome system does not require advanced biosafety levels. Furthermore, using minigenome system eliminates many potential confounding factors that may be present in coding regions of the genome. Use of the minigenome system is easier and more feasible than the full genome rescue of the virus. This study successfully shows the efficiency of the constructed rabies virus minigenome in expression of inserted gene.

Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest.

Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC50 value at 72 h (0.001 ± 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ± 0.037, 0.01 ± 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent.

An HIV-1 Mini Vaccine Induced Long-lived Cellular and Humoral Immune Responses.

Memory formation is the most important aspect of a vaccine which can guarantee long-lasting immunity and protection. The main aim of the present study was to evaluate the memory immune responses after immunization with a mini vaccine. Mice were immunized with human immunodeficiency virus-1 P24-Nef fusion peptide and then cellular and humoral immune responses were evaluated. In order to determine long-lived memory, immune responses were monitored for 20 weeks after final immunization. The results showed that the candidate vaccine induced proliferation and cytotoxic T lymphocyte responses and shifted cytokine patterns to T helper-1 profile. Evaluation of humoral immune responses also showed an increase in total peptide specific-IgG titer and a shift to IgG2a humoral response. Monitoring of immune responses at weeks 4, 12 and 20 after last immunization showed that immunologic parameters have been sustained for 20 weeks. Our findings support the notion that long-lived memory responses were achieved using a mini vaccine immunization.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells.

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples.

In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.

MiR-17-92 cluster: an apoptosis inducer or proliferation enhancer.

Study of the non-coding RNA roles in the regulation of adaptive immune responses through T cells could be the basis of novel therapeutic applications. MicroRNAs (miRNAs) are a class of short non-coding RNAs that control the cell's functions and destination. To investigate the role of miRNAs in T cell activation, herein the expressions of miR-17-92 cluster and its paralogs were studied in naïve CD4(+)T cells that were activated by anti-CD2, -CD3, -CD28 microbeads and induced with or without IL-2. Proliferation and apoptosis rate of the cultured cells were determined by BrdU incorporation assay (ELISA) and propidium iodide staining, respectively. In continuation the expressions of eight miRNAs of the mentioned clusters were analyzed quantitatively. In addition their potential targets were predicted using multiple algorithms; as a confirmation, the transcription of PIK3R3 (a putative target of modulated miRNAs) was evaluated. Stimulation index (SI) of activated cells was decreased on day 6; whereas, the IL-2 induced cells showed increase in SI in the assay time. Evaluation of eight members of the aforementioned cluster showed upregulation of miR-92a-2* (~15 times) in IL-2 un-induced (activated) cells relative to the IL-2 induced cells. In silico investigations revealed that the suggested miRNAs targeted genes that were involved in cell proliferation, survival, and apoptosis. Transcriptional analysis of PIK3R3 illustrated decrease in activated cells relative to IL-2 induced cells. According to our findings, it seems that multiple members of miR-17-92 families in activated CD4(+)T cells inhibited negative regulators of IL-2 such as DUSP, PTPN, and SOCS families after IL-2 induction. According to our findings, it seems that multiple genes of cell proliferation-related families such as MAPK, E2F, AKT, STAT, and JAK as well as PIK3R3 are inhibited by miR-17-92 cluster in activated cells. As FASL is a putative target of over-expressed miRNAs in activated cell, antigen-induced cell death (AICD) might be occurred in FASL-independent manner. Altogether this study suggested that clonal expansion through IL-2 signaling pathway does not depend on the members of miR-17-92 family; while, it appears that AICD in activated CD4(+)T cells without IL-2 induction is affected by these miRNA clusters.

Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis.

Development of a rapid and accurate quantification method for the detection of microRNAs (miRNAs) has been desired, in particular, when they are differently expressed in normal and pathological conditions. However, various methods for the quantification of small non-coding RNAs as well as miRNAs have been described. These methods mainly include hybridization-based approaches such as primer extension, northern blotting, microarray profiling, and reverse transcription (RT) PCR. Here, we developed a simple and rapid method based on stem-loop primer-based real-time PCR assay for sensitive and accurate detection of mature miRNAs. Initially, a miRNA-specific stem-loop RT primer is used for RT, which is followed by TaqMan real-time PCR assay using specific forward primer in combination with universal reverse primer and TaqMan probe. The assay has shown high sensitivity (≤50 copies/reaction) for miRNA detection in two breast cancer cell lines, MCF-7 and MDA-MB-231. This assay might be implicated as a rapid and cost effective method for the detection of small non-coding RNAs.

Detection of mucosal type human papillomavirus in cutaneous squamous cell carcinoma in Iran.

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer. The oncogenic role of human papilloma virus (HPV) in cutaneous SCC has been suggested by several studies performed on immunosuppresed patients. However, the role of mucosal type HPV in SCC patients with normal immunity has not been studied extensively. Sixty skin biopsies from immunocompetent SCC patients and 60 benign skin specimens were evaluated for mucosal type HPV DNA using polymerase chain reaction (PCR) and immunohistochemistry (IHC). Mucosal type HPV DNA was detected in 18 of 60 cases (30%) and in 7 of 60 controls (11.6%) using PCR. HPV immunostain was positive in 16 of 60 cases (26.6%) and in 15 of 60 controls (25%). Mixed infection with HPV 18, 11, 6 was found in half of the SCC cases. The most prevalent subtype was HPV 18 followed by HPV 6 and 11. The frequency of HPV DNA was significantly elevated in our cases compared to controls (P value <0.01, OR=16.8, 95% CI: 3.3-74.9). Our findings suggest an association of mucosal type HPV, especially HPV 18, with skin SCC in Iranian patients with normal immunity.

Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor.

Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

Human T-lymphotropic virus type 1 prevalence in northeastern Iran, Sabzevar: an epidemiologic-based study and phylogenetic analysis.

Human T-lymphotropic virus type 1 (HTLV-I) is an important global health problem in the world mainly in the endemic areas of HTLV-I infection. It was previously reported that Mashhad, in northeastern Iran, is a new endemic region of HTLV-I. The aim of this study was to examine the prevalence and phylogenetic analysis of HTLV-I in Sabzevar, located in the southeast of Mashhad. In this cross-sectional study 1445 individuals were selected by multistage cluster sampling. Serum samples were screened for anti-HTLV-I antibody using enzyme-linked immunosorbent assay (ELISA); all of the ELISA-positive samples were confirmed by polymerase chain reaction (PCR). Long terminal repeat (LTR) sequencing was carried out to determine the type of HTLV-I in Sabzevar. In the primary screening by ELISA, 26/1445 (1.8%) of those sampled were reactive for HTLV-I antibody. Twenty-four out of 26 samples were confirmed HTLV-I infection by PCR (24/1445). The overall prevalence of HTLV-I infection in Sabzevar is 1.66%. The prevalence of the virus infection in men and women was 2.42% (11/455) and 1.31% (13/989), respectively. Seroprevalence was associated with age, increasing significantly among those older than 30 years (p=0.015), and a history of surgery (p=0.002), imprisonment (p=0.018), and hospitalization (p=0.005). Three out of 24 positive HTLV-I samples were selected for sequencing and phylogenetic analysis of LTR. The results showed that HTLV-I in Sabzevar belonged to the cosmopolitan subtype. The present study showed Sabzevar is a new endemic area for HTLV-I infection. Our study emphasizes that systemic HTLV-I screening of blood donors in Sabzevar and other cities in Khorasan province is important and should be taken into account.

Epidermodysplasia verruciformis associated with plasmablastic lymphoma and hepatitis B virus infection.

Epidermodysplasia verruciformis is a rare genodermatosis characterized by inherited susceptibility to infection with certain papillomaviruses, which leads to the development of disseminated plane wart-like lesions. In some patients, lesions resembling pityriasis versicolor appear. Epidermodysplasia verruciformis has also been reported in immunosuppressed patients, most notably those with HIV infection. The affected patients are predisposed to development of skin and mucosal malignancies. We describe the rare occurrence of plasmablastic lymphoma in a patient with long lasting epidermodysplasia verruciformis and hepatitis B virus infection.

Mesenchymal stem cells as vehicles for targeted delivery of anti-angiogenic protein to solid tumors.

BACKGROUND: Inhibition of tumor-induced angiogenesis may restrict tumor growth and metastasis. Long-term systemic delivery of angiogenic inhibitors is associated with toxicity, as well as other severe side-effects. The utility of cells as vehicles for gene therapy to deliver therapeutic molecules has been suggested to represent an efficient approach. Mesenchymal stem cells (MSCs) exhibit a tropism to cancer tissue, and may serve as a cellular delivery vehicle and a local producer of anti-angiogenic agents. METHODS: In the present study, we attempted to assess production of the transgene, α1-antitrypsin (AAT), in lentivirus-transduced human MSCs and its cytotoxicity against human umbilical cord vein endothelial cells (HUVEC). The secreted protein from these effector cells was determined by an enzyme-linked immunosorbent assay. The cytotoxicity of hMSCs that overexpress the human AAT gene against HUVEC was evaluated with an apoptotic assay. RESULTS: Lentivirus-transduced hMSCs produced functional AAT and displayed much higher cytotoxicity against HUVEC than untransduced hMSCs. Moreover, AAT secreted from transduced hMSCs significantly inhibited HUVEC proliferation compared to untransduced hMSCs. The data obtained demonstrate for the first time that genetically modified hMSCs released abundant and functional AAT that caused obvious cytotoxicity to HUVEC. CONCLUSIONS: hMSC may serve as an effective platform for the targeted delivery of therapeutic proteins to cancer sites.