Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection.

Circular mRNA (cmRNA) is particular useful due to its high resistance to degradation by exonucleases, resulting in greater stability and protein expression compared to linear mRNA. T cell receptor (TCR)-engineered T cells (TCR-T) represent a promising means of treating viral infections and cancer. This study aimed to evaluate the feasibility and efficacy of cmRNA in antigen-specific-TCR discovery and TCR-T therapy. Using human cytomegalovirus (CMV) pp65 antigen as a model, we found that the expansion of pp65-responsive T cells was induced more effectively by monocyte-derived dendritic cells transfected with pp65-encoding cmRNA compared with linear mRNA. Subsequently, we developed cmRNA-transduced pp65-TCR-T (cm-pp65-TCR-T) that specifically targets the CMV-pp65 epitope. Our results showed that pp65-TCR could be expressed on primary T cells for more than 7 days. Moreover, both in vitro killing and in vivo CDX models demonstrated that cm-pp65-TCR-T cells specifically and persistently kill pp65-and HLA-expressing tumor cells, significantly prolonging the survival of mice. Collectively, our results demonstrated that cmRNA can be used as a more effective technical approach for antigen-specific TCR isolation and identification, and cm-pp65-TCR-T may provide a safe, non-viral, non-integrated therapeutic approach for controlling CMV infection, particularly in patients who have undergone allogeneic hematopoietic stem cell transplantation.

Expression profiles and characterization of microRNAs responding to chitin in Arthrobotrys oligospora.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it's target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

ADAMTS13 maintains cerebrovascular integrity to ameliorate Alzheimer-like pathology.

Blood-brain barrier (BBB) defects and cerebrovascular dysfunction contribute to amyloid-ß (Aß) brain accumulation and drive Alzheimer disease (AD) pathology. By regulating vascular functions and inflammation in the microvasculature, a disintegrin and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) plays a significant protective effect in atherosclerosis and stroke. However, whether ADAMTS13 influences AD pathogenesis remains unclear. Using in vivo multiphoton microscopy, histological, behavioral, and biological methods, we determined BBB integrity, cerebrovascular dysfunction, amyloid accumulation, and cognitive impairment in APPPS1 mice lacking ADAMTS13. We also tested the impact of viral-mediated expression of ADAMTS13 on cerebrovascular function and AD-like pathology in APPPS1 mice. We show that ADAMTS13 deficiency led to an early and progressive BBB breakdown as well as reductions in vessel density, capillary perfusion, and cerebral blood flow in APPPS1 mice. We found that deficiency of ADAMTS13 increased brain plaque load and Aß levels and accelerated cerebral amyloid angiopathy (CAA) by impeding BBB-mediated clearance of brain Aß, resulting in worse cognitive decline in APPPS1 mice. Virus-mediated expression of ADAMTS13 attenuated BBB disruption and increased microvessels, capillary perfusion, and cerebral blood flow in APPPS1 mice already showing BBB damage and plaque deposition. These beneficial vascular effects were reflected by increase in clearance of cerebral Aß, reductions in Aß brain accumulation, and improvements in cognitive performance. Our results show that ADAMTS13 deficiency contributes to AD cerebrovascular dysfunction and the resulting pathogenesis and cognitive deficits and suggest that ADAMTS13 may offer novel therapeutic opportunities for AD.

Alginate oligosaccharide modulates immune response, fat metabolism, and the gut bacterial community in grass carp (Ctenopharyngodon idellus).

Alginate oligosaccharide (AOS) is widely used in agriculture because of its many excellent biological properties. However, the possible beneficial effects of AOS and their underlying mechanisms are currently not well known in grass carp (Ctenopharyngodon idellus). Here, grass carp were fed diets supplemented with 5, 10, or 20 g/kg AOS for six weeks. HE and PAS staining showed that the diets of AOS significantly increased the number of goblet cells in the intestinal. According to transcriptome and quantitative real-time PCR (qRT-PCR) data, AOS-supplemented diets activated the expression of fat metabolism-related pathways and genes. The 16S rRNA sequencing results showed that supplementation with AOS affected the distribution and abundance of the gut bacterial assembly. qRT-PCR and activity assays revealed that the AOS diets significantly increased the antioxidant resistance in gut of grass carp, and down-regulated the expression of inflammatory and up-regulated anti-inflammatory cytokines. Finally, the Aeromonas hydrophila infection assay suggested that the mortality in the groups fed dietary AOS was slightly lower than that in the control. Therefore, supplementing the diet of grass carp with an appropriate amount of AOS can improve fat metabolism and immune responses and alter the intestinal bacterial community, which may help to fight bacterial infection.

ADAMTS13 controls vascular remodeling by modifying VWF reactivity during stroke recovery.

Angiogenic response is essential for ischemic brain repair. The von Willebrand factor (VWF)-cleaving protease disintegrin and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) is required for endothelial tube formation in vitro, but there is currently no in vivo evidence supporting a function of ADAMTS13 in angiogenesis. Here we show that mice deficient in ADAMTS13 exhibited reduced neovascularization, brain capillary perfusion, pericyte and smooth muscle cell coverage on microvessels, expression of the tight junction and basement membrane proteins, and accelerated blood-brain barrier (BBB) breakdown and extravascular deposits of serum proteins in the peri-infarct cortex at 14 days after stroke. Deficiency of VWF or anti-VWF antibody treatment significantly increased microvessels, perfused capillary length, and reversed pericyte loss and BBB changes in Adamts13-/- mice. Furthermore, we observed that ADAMTS13 deficiency decreased angiopoietin-2 and galectin-3 levels in the isolated brain microvessels, whereas VWF deficiency had the opposite effect. Correlating with this, overexpression of angiopoietin-2 by adenoviruses treatment or administration of recombinant galectin-3 normalized microvascular reductions, pericyte loss, and BBB breakdown in Adamts13-/- mice. The vascular changes induced by angiopoietin-2 overexpression and recombinant galectin-3 treatment in Adamts13-/- mice were abolished by the vascular endothelial growth factor receptor-2 antagonist SU1498. Importantly, treating wild-type mice with recombinant ADAMTS13 at 7 days after stroke markedly increased neovascularization and vascular repair and improved functional recovery at 14 days. Our results suggest that ADAMTS13 controls key steps of ischemic vascular remodeling and that recombinant ADAMTS13 is a putative therapeutic avenue for promoting stroke recovery.

Hemoglobin induces inflammation through NF-kB signaling pathway and causes cell oxidative damage in grass carp (Ctenopharyngodon idella).

Hemolytic disease in grass carp (C. idella) leads to hemolysis in vivo, releasing damage-related molecular patterns (DAMPs) hemoglobin (Hb; which is rapidly oxidized to Hb-Fe3+ and Hb-Fe4+) and generating a high level of reactive oxygen species (ROS) that cause oxidative damage. However, the effect of cell-free Hb on tissue cells of grass carp has yet to be elucidated. In this study, western blotting (WB) and immunofluorescence analysis (IFA) results showed that PHZ-induced hemolysis caused Hb and iron accumulation, increased the production of ROS and resulted in apoptosis in head kidney and middle kidney of the grass carp. Quantitative real-time PCR (qRT-PCR), WB, and IFA revealed that PHZ-induced hemolysis significantly upregulated the expression of inflammation-related genes through activation of the NF-κB signaling pathway. To further explore the effect of Hb, three forms of Hb (Hb, MetHb, and FerrylHb) were prepared. The incubation with the different forms of Hb and heme markedly upregulated the expression of cytokine genes through NF-κB signaling pathway, which was further confirmed by a specific inhibitor (caffeic acid phenethyl ester, CAPE). Flow cytometry analysis data showed that the stimulation of different forms of Hb and heme increased the production of ROS, and resulted in apoptosis. In summary, our data suggest that the excess cell-free Hb released during hemolysis modulates the inflammatory response through activation of the NF-κB signaling pathway and causes cell oxidative damage and apoptosis.

Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy.

The success of the two mRNA vaccines developed by Moderna and BioNTech during the COVID-19 pandemic increased research interest into the application of mRNA technologies. Compared with the canonical linear mRNA used in these vaccines, circular mRNA has been found to mediate more potent and durable protein expression and demands a simpler manufacturing procedure. However, the application of circular mRNA is still at the initiation stage, and proof of concept for its use as a future medicine or vaccine is required. In the current study, we established a novel type of circular mRNA, termed cmRNA, based on the echovirus 29-derived internal ribosome entry site element and newly designed homology arms and RNA spacers. Our results demonstrated that this type of circular mRNA could mediate strong and durable expression of various types of proteins, compared with typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of cmRNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and enhanced anti-programmed cell death protein 1 (PD-1) antibody-induced tumor repression in a syngeneic mouse model. This novel circular mRNA platform is thereby suitable for direct intratumoral administration for cancer therapy.

Transfusion of Resting Platelets Reduces Brain Hemorrhage After Intracerebral Hemorrhage and tPA-Induced Hemorrhage After Cerebral Ischemia.

BACKGROUND: Exacerbated blood-brain barrier (BBB) damage is related with tissue plasminogen activator (tPA)-induced brain hemorrhage after stroke. Platelets have long been recognized as the cellular orchestrators of primary haemostasis. Recent studies have demonstrated further that platelets are required for supporting intact mature blood vessels and play a crucial role in maintaining vascular integrity during inflammation. Therefore, we sought to investigate whether platelets could reduce tPA-induced deterioration of cerebrovascular integrity and lead to less hemorrhagic transformation. METHODS: Mice were subjected to models of collagenase-induced intracerebral hemorrhage (ICH) and transient middle cerebral artery (MCA) occlusion. After 2 h of MCA occlusion, tPA (10 mg/kg) was administered as an intravenous bolus injection of 1 mg/kg followed by a 9 mg/kg infusion for 30 min. Immediately after tPA treatment, mice were transfused with platelets. Hemorrhagic volume, infarct size, neurological deficit, tight junction and basal membrane damages, endothelial cell apoptosis, and extravascular accumulation of circulating dextran and IgG, and Evans blue were quantified at 24 h. RESULTS: Platelet transfusion resulted in a significant decrease in hematoma volume after ICH. In mice after ischemia, tPA administration increased brain hemorrhage transformation and this was reversed by resting but not activated platelets. Consistent with this, we observed that tPA-induced brain hemorrhage was dramatically exacerbated in thrombocytopenic mice. Transfusion of resting platelets ameliorated tPA-induced loss of cerebrovascular integrity and reduced extravascular accumulation of circulating serum proteins and Evans blue, associated with improved neurological functions after ischemia. No changes were found for infarct volume. Inhibition of platelet receptor glycoprotein VI (GPVI) blunted the ability of platelets to attenuate tPA-induced BBB disruption and hemorrhage after ischemia. CONCLUSION: Our findings demonstrate the importance of platelets in safeguarding BBB integrity and suggest that transfusion of resting platelets may be useful to improve the safety of tPA thrombolysis in ischemic stroke.

Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13.

Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA.

von Willebrand factor contributes to poor outcome in a mouse model of intracerebral haemorrhage.

Spontaneous intracerebral haemorrhage (ICH) is the most devastating stroke subtype and has no proven treatment. von Willebrand factor (VWF) has recently been demonstrated to promote inflammation processes. The present study investigated the pathophysiological role of VWF after experimental ICH. Functional outcomes, brain edema, blood-brain barrier (BBB) permeability, cerebral inflammation and levels of intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinase-9 (MMP-9) were measured in a mouse model of ICH induced by autologous blood injection. We show that VWF were increased in the plasma and was accumulated in the perihematomal regions of mice subjected to ICH. Injection of VWF resulted in incerased expression of proinflammatory mediators and activation of ICAM-1 and MMP-9, associated with elevated myeloperoxidase, recruitment of neutrophils and microglia. Moreover, mice treated with VWF showed dramatically decreased pericyte coverage, more severe BBB damage and edema formation, and neuronal injury was increased compared with controls. In contrast, blocking antibodies against VWF reduced BBB damage and edema formation and improved neurological function. Together, these data identify a critical role for VWF in cerebral inflammation and BBB damage after ICH. The therapeutic interventions targeting VWF may be a novel strategy to reduce ICH-related injury.