Development of nanoparticle vaccines utilizing designed Fc-binding homo-oligomers and RBD-Fc of SARS-CoV-2.

The Fc-fused receptor binding domain (RBD-Fc) vaccine for SARS-CoV-2 has garnered significant attention for its capacity to provide effective and specific immune protection. However, its immunogenicity is limited, highlighting the need for improvement in clinical application. Nanoparticle delivery has been shown to be an effective method for enhancing antigen immunogenicity. In this study, we developed bivalent nanoparticle recombinant protein vaccines by assembling the RBD-Fc of SARS-CoV-2 and Fc-binding homo-oligomers o42.1 and i52.3 into octahedral and icosahedral nanoparticles. The formation of RBD-Fc nanoparticles was confirmed through structural characterization and cell binding experiments. Compared to RBD-Fc dimers, the nanoparticle vaccines induced more potent neutralizing antibodies (nAb) and stronger cellular immune responses. Therefore, using bivalent nanoparticle vaccines based on RBD-Fc presents a promising vaccination strategy against SARS-CoV-2 and offers a universal approach for enhancing the immunogenicity of Fc fusion protein vaccines.

Recent advances in mesenchymal stem cell therapy for myocardial infarction.

Myocardial infarction refers to the ischemic necrosis of myocardium, characterized by a sharp reduction or interruption of blood flow in the coronary arteries due to the coronary artery occlusion, resulting in severe and prolonged ischemia in the corresponding myocardium and ultimately leading to ischemic necrosis of the myocardium. Given its high risk, it is considered as one of the most serious health threats today. In current clinical practice, multiple approaches have been explored to diminish myocardial oxygen consumption and alleviate symptoms, but notable success remains elusive. Accumulated clinical evidence has showed that the implantation of mesenchymal stem cell for treating myocardial infarction is both effective and safe. Nevertheless, there persists controversy and variability regarding the standardizing MSC transplantation protocols, optimizing dosage, and determining the most effective routes of administration. Addressing these remaining issues will pave the way of integration of MSCs as a feasible mainstream cardiac treatment.

Fluorescence quenching-based immunological probe for ticagrelor monitoring.

Introduction: Ticagrelor is extensively utilized for the treatment of acute coronary syndromes (ACS), but its platelet aggregation inhibitory effects can potentially result in tissue bleeding, posing a serious risk to patients' lives. Methods: In this study, we developed highly sensitive full length anti-ticagrelor Quenchbodies (Q-bodies) for fast monitoring of ticagrelor both in solution and serum for the first time. Ticagrelor coupled with N- hydroxysuccinimide (Ticagrelor-NHS) ester was also designed and synthesized for interaction and biological activity detection. Results: Both ATTO-labeled MEDI2452 (2452A) Q-body and TAMRA-labeled IgG 152 (152T) Q-body demonstrated efficient detection of ticagrelor and its active metabolite (TAM). The 2452A Q-body exhibited a broader detection range, while the 152T Q-body displayed a lower limit of detection (LOD). Under physiological conditions (Ticagrelor:TAM, 3:1), the concentration of ticagrelor was further measured, yielding LOD values of 4.65 pg/mL and 2.75 pg/mL for the two Q-bodies, with half-maximal effect concentrations of 8.15 ng/mL and 3.0 ng/mL, respectively. Discussion: Compared with traditional liquid chromatography-mass spectrometry (LC-MS) methods, anti-ticagrelor Q-bodies have higher sensitivity and detection speed. It enabled the completion of analysis within 3 min, facilitating rapid preoperative detection of blood drug concentration in ACS to determine the feasibility of surgery and mitigate the risk of intraoperative and postoperative hemorrhage. The swift detection of ticagrelor holds promise for enhancing individualized drug administration, preventing adverse reactions, and providing preoperative guidance.

Cyclic thrombocytopenia associated with estradiol: a case report.

OBJECTIVES: Cyclic thrombocytopenia (CTP) is a rare blood disorder characterized by periodic fluctuations in platelet counts. CTP usually appears in pre-menopausal women, and these fluctuations of platelets are in phase with the menstrual cycle. CTP is a heterogeneous disease, and the pathogenic mechanism is still unclear. Therefore, it harbors great significance for exploring the association of fluctuations in platelet counts with hormonal-cycle. MATERIALS: Firstly, we washed human platelets from healthy volunteers following the Declaration of Helsinki. Flow cytometer was employed to measure the mitochondrial inner transmembrane potential (ΔΨm) depolarization, PS exposure, P-selectin expression, and GPIIb/IIIa activation in platelets. In addition, western blot detected the related protein expression. The corresponding assay kit measured the caspase-3 and PDE3A activity. Finally, flow cytometry determined mouse platelets labeled with calcein. RESULTS: We find a reverse relationship between the platelet count and serum estradiol (E2) level in a CTP patient. We demonstrated that E2 induces platelet apoptosis in vitro and platelet clearance in vivo. We further discovered that E2 activates phosphodiesterase 3A, which inhibits protein kinase A (PKA), leading to PKA-mediated platelet apoptosis. Activation of PKA protected platelets from E2-induced thrombocytopenia and elevated the number of mice circulatory platelets. CONCLUSIONS: We find that E2 induces platelet apoptosis and clearance through PDE3A-mediated PKA inhibition. Activation of PKA rescues E2-induced thrombocytopenia in mice. Thus, our study reveals a pathogenesis of E2-related CTP and suggests promising therapeutic strategies for the disease.

The cusp catastrophe warning model for easily weathered granite considering energy accumulation and dissipation.

To solve the problem of destabilization catastrophe damage to easily weathered granite (EWG) which is easy to soften and disintegrate after absorbing water, there is an urgent need to study the destabilization catastrophe warning of EWG. The destabilization and catastrophe damage characteristics of EWG were analyzed from the perspective of energy accumulation and dissipation. First, the relevant data of EWG were obtained by uniaxial compression and acoustic emission (AE) test. The abrupt characteristics of the evolution of the elastic strain energy (ESE) curve are discussed. Meanwhile, the cusp catastrophe theory is introduced, and the cusp catastrophe warning model of destabilizing mutation of EWG is constructed based on the strain sequence of ESE. Then, the mutation interval, early warning point, and the early warning interval of EWG under uniaxial compression are defined. Finally, the reasonableness of the cusp catastrophe warning model was verified by the precursor information characteristics of AE parameters. The results of the study can be helpful for the early warning and prevention of destabilizing mutations in EWG.

Actin polymerization regulates glycoprotein Ibα shedding.

Glycoprotein (GP) Ibα shedding mediated by ADAM17 (a disintegrin and metalloproteinase 17) plays an important role in negatively regulating platelet function and thrombus formation. However, the mechanism of GPIbα shedding remains elusive. Here, we show that jasplakinolide (an actin-polymerizing peptide)-induced actin polymerization results in GPIbα shedding and impairs platelet function. Thrombin and A23187-induced GPIbα shedding is increased by jasplakinolide; in contrast, GPIbα shedding is reduced by a depolymerization regent (cytochalasin B). We find that actin polymerization activates calpain leading to filamin A hydrolyzation. We further demonstrate that the interaction of filamin A with the cytoplasmic domain of GPIbα plays a critical role in regulating actin polymerization-induced GPIbα shedding. Taken together, these data demonstrate that actin polymerization regulates ADAM17-mediated GPIbα shedding, suggesting a novel strategy to negatively regulate platelet function.

Carbamazepine Induces Platelet Apoptosis and Thrombocytopenia Through Protein Kinase A.

Carbamazepine is extensively used worldwide to treat a wide range of disorders such as epilepsy, peripheral neuralgia and bipolar disorder. Thrombocytopenia and hemorrhage have been identified in multiple carbamazepine-treated patients. However, the underlying mechanism remains poorly understood. Here, we show that platelets undergo apoptosis after carbamazepine treatment. The apoptotic platelets induced by carbamazepine are rapidly removed in vivo, which accounts for thrombocytopenia. We found that carbamazepine treatment attenuates the phosphorylation level of bcl-xl/bcl-2-associated death promoter (BAD), vasodilator-associated stimulated phosphoprotein (VASP) and GPIbß in platelets, indicating an inhibition effect on protein kinase A (PKA). We further demonstrated that carbamazepine reduced PKA activity through PI3K/Akt/PDE3A signaling pathway. Pharmacological activation of PKA or inhibition of PI3K/Akt/PDE3A protects platelets from apoptosis induced by carbamazepine. Importantly, PDE3A inhibitors or PKA activator ameliorates carbamazepine-mediated thrombocytopenia in vivo. These findings shed light on a possible mechanism of carbamazepine-induced thrombocytopenia, designating PDE3A/PKA as a potential therapeutic target in the treatment of carbamazepine-induced thrombocytopenia.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Adenovirus-mediated interleukin 21 gene transfer enhances antitumor immunity and reduces tumorigenicity of Hepa1-6 in mice.

In the present study, adenovirus-mediated interleukin 21 (Ad5-IL-21-EGFP) gene expression was induced in Hepa1-6 cells to investigate whether IL-21 was capable of enhancing antitumor immunity and reducing tumorigenicity of Hepa1-6 in a mouse model. Mice were inoculated intradermally into the right flank with Hepa1-6 cells or Hepa1-6 cells infected with Ad5 or Ad5-IL-21. Four weeks later, the mice were sacrificed humanely, and the tumor volume, tumor weight and mouse spleen index were measured. The levels of IL-21, IL-4 and interferon (IFN)-γ levels in mouse serum and tumor tissues were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Cell counting kit-8 (CCK-8) assay was used to detect the killing ability of spleen T cells and natural killer (NK) cells, and the proliferation ability of T cells. The expression of IL-21 was confirmed by reverse transcription-polymerase chain reaction, western blot analysis and ELISA assay in Ad5-IL-21-EGFP-infected Hepa1-6 cells. The overexpression of IL-21 significantly reduced the tumorigenicity of Hepa1-6 cells. The tumor volumes and tumor weights in Ad5-IL-21-Hepa1-6 mice were much smaller than those in the Ad5-Hepa1-6 group and Hepa1-6 wild-type group. The immunohistochemistry and ELISA assay demonstrated that IL-21 and IFN-γ levels were much higher while the IL-4 level was much lower in the Ad5-IL-21-Hepa1-6 group than in the other two groups. CCK-8 assay revealed that the killing ability of NK cells and T cells, and the proliferation ability of T cells in Ad5-IL-21-Hepa1-6 mice were higher than in the other two groups; the spleen index of Ad5-IL-21-Hepa1-6 mice was also higher than in the other groups. The data had a significant difference (P<0.01). In conclusion, IL-21 reduces tumorigenicity of Hepa1-6 by a mechanism involving enhanced activation of cell-mediated immunity in tumor-bearing mice.

Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice.

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1ß in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.

[The expression of human IL-1ß gene containing human erythropoietin signal peptide in HepG2 cells].

OBJECTIVE: To construct two lentiviruses secreting human IL-1ß through either classical or nonclassical pathway and analyze their expressions in HepG2 cells after packaging lentiviruses and infecting hepatoma carcinoma HepG2 cells. METHODS: Human full-length IL-1ß gene and chimeric gene containing human erythropoietin(EPO) signal peptide and mature IL-1ß protein coding sequence were respectively amplified from pIRES2-EGFP-proIL-1ß and pIRES2-EGFP-epoIL-1ß using PCR. The sequences were subsequently cloned into lentiviral expression vector pLenti6/V5 to construct pLenti6/V5-proIL-1ß, which expressed IL-1ß through nonclassical pathway, and pLenti6/V5-epoIL-1ß, which expressed IL-1ß through signal-peptide mediated classical pathway. Lentiviruses expressing human IL-1ß through either classical or nonclassical pathway were packaged in HEK293T cells using a three-plasmid packaging system, and then these viruses were used to infect HepG2 cells. The level of IL-1ß in both cytoplasm and culture supernatant were detected by sandwich ELISA and Western blotting. RESULTS: pLenti6/V5-proIL-1ß expressing human full-length IL-1ß gene and pLenti6/V5-epoIL-1ß expressing human EPO signal peptide and mature IL-1ß gene were successfully constructed and confirmed through enzymatic assay and DNA sequencing. The lentiviruses expressing IL-1ß through different pathways were prepared using a three-plasmid packaging system in HEK293T cells. Compared with the cells infected with control virus, levels of supernatant and cytoplasmic IL-1ß in the cells infected with two lentiviruses expressing IL-1ß through different pathways were markedly elevated (P<0.01). However, level of mature IL-1ß in supernatant of HepG2/epoIL-1ß cells was much higher than that of HepG2/proIL-1ß cells, while total IL-1ß level in cytoplasm of HepG2/proIL-1ß cells was significantly higher than that in HepG2/epoIL-1ß cells. CONCLUSION: Both classical and nonclassical pathway secretion vectors could express human IL-1ß in HepG2 cells, but EPO signal-peptide mediated classical pathway secreted much higher mature IL-1ß than that of nonclassical pathway.

[IL-1ß can promote proliferation and migration of Hepa1-6 cells and impair IFNα-induced cell growth inhibition in vitro].

AIM: To analyze the effect of recombinant IL-1ß on proliferation, migration, and the effect on IFNα induced cell growth inhibtion. METHODS: The vector pLIVE-mIL-1ß was transfected into Hepa1-6 cells mediated by transIT-LT1. Gene expression level of IL-1ß was analyzed by RT-PCR and Sandwich ELISA. Cell migration was assessed using wound healing assay. RESULTS: IL-1ß significantly stimulated proliferation and migration of Hepa1-6 cells. However, expression of IL-1ß significantly down-regulated growth inhibition inducecd by IFNα. CONCLUSION: The recombinant vector could stably express IL-1ß and promote in vitro proliferation, migration, and impair IFNα-induced cell growth inhibition.

[Expression of recombinant mouse IL-1beta significantly down-regulates NK cell mediated cytolysis [corrected] against H22 hepatoma cells].

AIM: To investigate the effect of the expression of recombinant IL-1beta in H22 hepatoma cells on its response to NK cell mediated cytotoxicity. METHODS: BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes (PBMCs). IL-1beta gene (843 bp) was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into pIRES2-EGFP to construct the recombinant pIRES2-EGFP-mIL-1beta expression vector which was verified by PCR, restriction enzyme assay (Xho I and EcoR I) and DNA sequencing. Then the purified pIRES2-EGFP-mIL-1beta plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1beta was detected by RT-PCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay. RESULTS: After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1beta gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by pIRES2-EGFP to create pIRES2-EGFP-mIL-1beta expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then pIRES2-EGFP-mIL-1beta was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1beta expression vector. In a 4-hour based MTT assay, IL-1beta in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with pIRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40:1. CONCLUSION: The expression of proinflammatory cytokine IL-1beta can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.

[IL-1beta antisense RNA enhanced sensitivity of HepG2 cells to NK cell mediated cytotoxicity].

AIM: To investigate the inhibitory effect of IL-1beta antisense RNA on the sensitivity of HepG2 cells to the NK cell mediated cytotoxicity. METHODS: Two gene segments of IL-1beta [IL-1beta1(17-331, 315 bp) and IL-1beta2(246-505, 260 bp)] were selected for antisense RNA. Total RNA was extracted from PBMC of a healthy donor treated with LPS. IL-1beta1 and IL-1beta2 were prepared by RT-PCR. PCR products were cloned into pMD18-T-simple vector and then sub-cloned to construct the pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 antisense RNA expression vectors. HepG2 cells were transfected by jetPEI, the expression of antisense RNA in HepG2 cells was assayed by RT-PCR, level of IL-1beta was analyzed by intracellular staining. The response of HepG2 cells to NK-92 cells was assessed by MTT assay. RESULTS: Two gene fragments of 260 bp and 315 bp products were obtained by RT-PCR. The purified gene fragments were cloned to construct pMD18 T-IL-1beta1 and pMD18 T-IL-1beta2 which were verified by PCR, restriction enzyme assay (Xho I) and DNA sequencing. The PCR products using Pfu DNA polymerase from cloning vectors were sub-cloned to create the antisense RNA expression vectors of pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 which were confirmed by PCR, restriction enzyme assay (Pst I) and DNA sequencing. When transfected into HepG2 cells, HepG2 cells expressed high level of antisense RNA, and simultaneously expression of IL-1beta was markedly suppressed which rendered HepG2 cells to be more sensitive to NK-92 cell mediated cytotoxicity compared with the cells transfected by pcDNA3.0. The cytolytic activity of NK-92 cells to HepG2 cells increase about 20% at the effector to target ratio of 10:1. CONCLUSION: Inhibiting of proinflammatory cytokine IL-1beta can reduce the sensitivity of hepatoma cells to the NK cell mediated cytolysis which provide an useful way of rendering NK cell activity against hepatoma.