-
Microorganisms May 2024The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study,...
The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study, we immunized 6-week-old female C57BL/6J mice via subcutaneous injection with 10 μg of purified C129R protein. Humoral and cellular immune effects were assessed using ELISA, flow cytometry, and ELISpot assays. Additionally, 19 peptides of the C129R protein were synthesized and screened for the use of bioinformatics. Positive T-cell epitopes were screened using ELISpot. The results indicated a higher proportion of CD4+ and CD8+ T lymphocytes in immunized mice compared to control mice. ELISA analysis revealed a serum titer of approximately 1:1, 638, 400 in the experimental group of mice. Additionally, peptides C11, C14, C16, and C18 from the C129R protein were able to activate mice spleen lymphocytes to produce IFN-γ. These findings suggest that the C129R protein significantly enhances both humoral and cellular immunity in immunized mice. Moreover, peptides C11, C14, C16, and C18 may serve as potential T-cell epitopes for the C129R protein. These results lay the groundwork for the further exploration of ASFV C129R protein and the identification of novel ASF vaccine antigens.
PubMed: 38930438
DOI: 10.3390/microorganisms12061056 -
International Journal of Molecular... Jun 2024Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for...
Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.
Topics: Myosin Heavy Chains; Humans; Transfection; Cell Line; Animals; Mice; Cardiac Myosins
PubMed: 38928453
DOI: 10.3390/ijms25126747 -
International Journal of Molecular... Jun 2024Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It...
Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.
Topics: Humans; Virus Replication; Oncolytic Viruses; RNA, Viral; Adenoviridae; Animals; Oncolytic Virotherapy; Mice; Cell Line, Tumor; Neoplasms; Female
PubMed: 38928259
DOI: 10.3390/ijms25126551 -
International Journal of Molecular... Jun 2024Clinical treatment options to combat Encephalopathy of Prematurity (EoP) are still lacking. We, and others, have proposed (intranasal) mesenchymal stem cells (MSCs) as a...
Clinical treatment options to combat Encephalopathy of Prematurity (EoP) are still lacking. We, and others, have proposed (intranasal) mesenchymal stem cells (MSCs) as a potent therapeutic strategy to boost white matter repair in the injured preterm brain. Using a double-hit mouse model of diffuse white matter injury, we previously showed that the efficacy of MSC treatment was time dependent, with a significant decrease in functional and histological improvements after the postponement of cell administration. In this follow-up study, we aimed to investigate the mechanisms underlying this loss of therapeutic efficacy. Additionally, we optimized the regenerative potential of MSCs by means of genetic engineering with the transient hypersecretion of beneficial factors, in order to prolong the treatment window. Though the cerebral expression of known chemoattractants was stable over time, the migration of MSCs to the injured brain was partially impaired. Moreover, using a primary oligodendrocyte (OL) culture, we showed that the rescue of injured OLs was reduced after delayed MSC coculture. Cocultures of modified MSCs, hypersecreting IGF1, LIF, IL11, or IL10, with primary microglia and OLs, revealed a superior treatment efficacy over naïve MSCs. Additionally, we showed that the delayed intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, improved myelination and the functional outcome in EoP mice. In conclusion, the impaired migration and regenerative capacity of intranasally applied MSCs likely underlie the observed loss of efficacy after delayed treatment. The intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, is a promising optimization strategy to prolong the window for effective MSC treatment in preterm infants with EoP.
Topics: Animals; Mesenchymal Stem Cells; Mice; Mesenchymal Stem Cell Transplantation; Secretome; Disease Models, Animal; Oligodendroglia; Humans; Coculture Techniques; Microglia; Mice, Inbred C57BL
PubMed: 38928201
DOI: 10.3390/ijms25126494 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To investigate the effects of the serine/threonine kinase family member 1 () gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the...
OBJECTIVE
To investigate the effects of the serine/threonine kinase family member 1 () gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway.
METHODS
Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 μmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of mRNA and added with CoA1 at a concentration of 20 μmol/L). After culture for 24 h, the expressions of mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate.
RESULTS
The mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia ( < 0.05). Compared with U937 group, mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G /M phase proportions were decreased in Si-PIM1 group (all < 0.05), while p27, Caspase-3 protein, G/G phase proportion and apoptosis rate were increased (all < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group ( >0.05).
CONCLUSION
Knockdown of gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.
Topics: Humans; Janus Kinase 2; Proto-Oncogene Proteins c-pim-1; STAT3 Transcription Factor; Cell Proliferation; Apoptosis; Signal Transduction; Leukemia, Myeloid, Acute; U937 Cells
PubMed: 38926951
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.003 -
The Science of the Total Environment Jun 2024The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as...
The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns.
PubMed: 38925381
DOI: 10.1016/j.scitotenv.2024.174238 -
International Immunopharmacology Jun 2024Hepatic ischemia-reperfusion (IR) injury is a major complication of liver transplantation and gravely affects patient prognosis. Icaritin (ICT), the primary plasma...
BACKGROUND
Hepatic ischemia-reperfusion (IR) injury is a major complication of liver transplantation and gravely affects patient prognosis. Icaritin (ICT), the primary plasma metabolite of icariin (ICA), plays a critical role in anti-inflammatory and immunomodulatory processes. However, the role of ICT in hepatic IR injury remains largely undefined. In this study, we aimed to elucidate the role of ICT in hepatic IR injury.
METHODS
We established hepatic IR injury models in animals, as well as an oxygen-glucose deprivation/reperfusion (OGD/R) cell model. Liver injury in vivo was assessed by measuring serum alanine aminotransferase (ALT) levels, necrotic areas by liver histology and local hepatic inflammatory responses. For in vitro analyses, we implemented flow-cytometric and western blot analyses, transmission electron microscopy, and an mRFP-GFP-LC3 adenovirus reporter assay to assess the effects of ICT on OGD/R injury in AML12 and THLE-2 cell lines. Signaling pathways were explored in vitro and in vivo to identify possible mechanisms underlying ICT action in hepatic IR injury.
RESULTS
Compared to the mouse model group, ICT preconditioning considerably protected the liver against IR stress, and diminished the levels of necrosis/apoptosis and inflammation-related cytokines. In additional studies, ICT treatment dramatically boosted the expression ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR proteins in hepatic cells following OGD/R damage. We also applied LY294002 (a PI3K inhibitor) and RAPA (rapamycin, an mTOR inhibitor), which blocked the protective effects of ICT in hepatocytes subjected to OGD/R.
CONCLUSION
This study indicates that ICT attenuates ischemia-reperfusion injury by exerting anti-inflammation, anti-oxidative stress, and anti-autophagy effects, as demonstrated in mouse livers. We thus posit that ICT could have therapeutic potential for the treatment of hepatic IR injury.
PubMed: 38924868
DOI: 10.1016/j.intimp.2024.112533 -
PLoS Pathogens Jun 2024Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused...
Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan virus resulted in 164 cases with 55 deaths. In 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea and Tanzania resulting in over 49 confirmed or suspected cases; 41 of which were fatal. There are no clearly defined correlates of protection against these VHF, impeding targeted vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Ebola virus, Sudan virus, Marburg virus) and an arenavirus (Lassa virus). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19/AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Ebola virus, Sudan virus and Marburg virus challenges in a small animal model.
PubMed: 38924060
DOI: 10.1371/journal.ppat.1012262 -
Nano Letters Jun 2024Enhancing the antitumor immune response and targeting ability of oncolytic viruses will improve the effect of tumor immunotherapy. Through infecting neural stem cells...
Enhancing the antitumor immune response and targeting ability of oncolytic viruses will improve the effect of tumor immunotherapy. Through infecting neural stem cells (NSCs) with a capsid dual-modified oncolytic adenovirus (CRAd), we obtained and characterized the "oncolytic extracellular vesicles" (CRAdEV) with improved targeted infection and tumor killing activity compared with CRAd. Both and studies revealed that CRAdEV activated innate immune cells and importantly enhanced the immunomodulatory effect compared to CRAd. We found that CRAdEV effectively increased the number of DCs and activated CD4 and CD8 T cells, significantly increased the number and activation of B cells, and produced higher levels of tumor-specific antibodies, thus eliciting enhanced antitumor activity compared with CRAd in a B16 xenograft immunocompetent mice model. This study provides a novel approach to oncolytic adenovirus modification and demonstrates the potential of "oncolytic extracellular vesicles" in antitumor immunotherapy.
PubMed: 38922640
DOI: 10.1021/acs.nanolett.4c02279 -
Pathogens (Basel, Switzerland) Jun 2024Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in...
Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site ( < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.
PubMed: 38921784
DOI: 10.3390/pathogens13060486