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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.
RESUMO
Background: Colorectal Cancer (CRC) represents a significant global health challenge, and its progression, resistance to therapy, and metastasis are strongly influenced by the tumor microenvironment, including factors like hypoxia. This study explores the impact of High Mobility Group Box 1 (HMGB1) overexpression on CRC cell migration, while identifying potential genes associated with this process. Methods: To explore this, we developed oncolytic virotherapy, resulting in HSVHMGB1, an oncolytic Herpes simplex virus that expresses HMGB1. HMGB1 is known its role in cancer progression, particularly in the context of cancer cell migration. Results: Contrary to expectations, our scratch assays indicated that HSV-HMGB1 did not significantly induce migration in CRC cells, suggesting that HMGB1 might not directly contribute to this process. Employing microarray analysis, we investigated gene expression changes linked to CRC cell migration, leading to construction of a Protein-Protein Interaction (PPI) network. This network revealed the presence of hub proteins, including as NDRG1, LGALS1, and ANGPTL4, which are recognized for their roles in cancer cell migration. The differential expression of these genes under hypoxic conditions was further validated using quantitative RT-PCR, aligning with the findings from our microarray data. Conclusion: Our findings emphasize the complex regulation of CRC cell migration, and provides valuable insights into potential molecular mechanisms and pathways. These findings have implications for further research into cancer progression and the development of therapeutic strategies.
RESUMO
AIMS: Despite the existence of effective preventive vaccines for human papillomavirus (HPV), therapeutic vaccines that trigger cell-mediated immune responses are required to treat established infections and malignancies. The aim of this study was to evaluate the therapeutic potency of HPV-16 E7 deoxyribonucleic acid (DNA) vaccine alone and with interleukin (IL)-18. METHODS: In vitro expressions of IL-18 were performed on human embryonic kidney 293 cells and confirmed it by Western blotting methods. DNA vaccine was available from a previous study. A total of 45 female C57BL/6 mice divided into five groups (DNA vaccine, DNA vaccine adjuvanted with IL-18, pcDNA3.1, and phosphate buffer saline) were inoculated with murine tissue culture-1 cell line of HPV related carcinoma, expressing HPV-16 E6/E7 antigens. They were then immunized subcutaneously twice at a seven-day interval. The antitumor and antigen specific-cellular immunity were assessed by lymphocyte proliferation (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: MTT) assay, lactate dehydrogenase release assay, IL-4 assay and interferon-gamma (IFN-γ) assay. Tumor size was followed for 62 days. RESULTS: MTT assay, which measures the lymphocyte proliferation in response to the specific antigen, increased in the co-administration and the DNA vaccine groups as compared to control and genetic adjuvant groups (p<0.001). The mice immunized with the co-administration generated significantly more IFN-γ and IL-4 than other immunized mice (p<0.001). Reduction of the tumor size in the co-administration and the DNA vaccine groups was significantly more pronounced than in the control and genetic adjuvant groups (p<0.001), but no statistically significant difference between DNA vaccine and co-administration groups (p=0.15) occurred. CONCLUSIONS: IL-18 as a genetic adjuvant and E7 DNA vaccine alone enhanced immune responses in mouse model systems against cervical cancer. However, using of IL-18 as a genetic adjuvant with E7 DNA vaccine had no significant synergistic effect on the immune responses in vivo.
OBJETIVOS: Apesar da existência de vacinas preventivas eficazes contra o papilomavírus humano (HPV), são necessárias vacinas terapêuticas que desencadeiem respostas imunes mediadas por células para tratar infecções e malignidades estabelecidas. O objetivo deste estudo foi avaliar a potência terapêutica da vacina de ácido desoxirribonucleico (DNA) HPV-16 E7 isolada e com interleucina (IL)-18. MÉTODOS: Expressões in vitro de IL-18 foram realizadas em células renais embrionárias humanas 293 e confirmadas por Western blotting. A vacina de DNA foi disponibilizada em um estudo anterior. Um total de 45 camundongos fêmeas C57BL/6 divididos em cinco grupos (vacina de DNA, vacina de DNA adjuvada com IL-18, pcDNA3.1 e solução salina tamponada com fosfato) e foram inoculados com linhagem murina-1 de carcinoma relacionado ao HPV, expressando antígenos E6 / E7 do HPV-16. Os animais foram então imunizados por via subcutânea duas vezes no intervalo de sete dias. A imunidade antitumoral e antígeno-celular específica foi avaliada pela proliferação de linfócitos (ensaio de brometo de 3- [4,5-dimetiltiazol-2-il] -2,5-difeniltetrazólio: MTT), ensaio de liberação de lactato desidrogenase, ensaio de IL-4 e ensaio de interferon-gama [IFN-γ]. O tamanho do tumor foi seguido por 62 dias. RESULTADOS: O ensaio MTT, que mede a proliferação de linfócitos em resposta ao antígeno específico, aumentou nos grupos de coadministração e de vacina de DNA em comparação aos grupos controle e adjuvante genético (p <0,001). Os camundongos imunizados com a coadministração geraram significativamente mais IFN-γ e IL-4 do que os outros camundongos imunizados (p<0,001). A redução do tamanho do tumor nos grupos de coadministração e de vacina de DNA foi significativamente mais acentuada do que nos grupos controle e adjuvante genético (p<0,001), mas não houve nenhuma diferença estatisticamente significativa entre os grupos vacina de DNA e coadministração (p=0,15). CONCLUSÕES: A IL-18 como adjuvante genético e a vacina de DNA E7 aumentaram as respostas imunes em sistemas modelo de camundongos contra o câncer cervical. No entanto, o uso de IL-18 como adjuvante genético com a vacina de DNA E7 não teve efeito sinérgico significativo nas respostas imunes in vivo.