Fulminant myocarditis induced by SARS-CoV-2 infection without severe lung involvement: insights into COVID-19 pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection often leads to pulmonary complications. Cardiovascular sequelae, including myocarditis and heart failure, have also been reported. Here, the study presents two fulminant myocarditis cases infected by SARS-CoV-2 exhibiting remarkable elevation of cardiac biomarkers without significant pulmonary injury, as determined by imaging examinations. Immunohistochemical staining reveals viral antigen within cardiomyocytes, indicating that SARS-CoV-2 could directly infect myocardium. The full viral genomes from respiratory, anal, and myocardial specimens are obtained via next-generation sequencing. Phylogenetic analyses of the whole genome and spike gene indicate that viruses in the myocardium/pericardial effusion and anal swabs are closely related and cluster together yet diverge from those in the respiratory samples. In addition, unique mutations are found in the anal/myocardial strains compared to the respiratory strains, suggesting tissue-specific virus mutation and adaptation. These findings indicate genetically distinct SARS-CoV-2 variants have infiltrated and disseminated within myocardial tissues, independent of pulmonary injury, and point to different infection routes between the myocardium and respiratory tract, with myocardial infections potentially arising from intestinal infection. These findings highlight the potential for systemic SARS-CoV-2 infection and the importance of a thorough multi-organ assessment in patients for a comprehensive understanding of the pathogenesis of COVID-19.

Biomimetic exosomal vesicles loaded with siRNA improves antitumor immune responses by inhibiting the secretion of tumor-derived exosome PD-L1.

Tumor-derived exosome PD-L1 exhaustsTcells and permits tumor cells to evade immune surveillance; thus, the inhibition of ExoPD-L1 secretion can significantly enhance the clinical efficacy of PD-L1 antibody. In this study, we combined exosome membrane, apoA1 and phospholipid into biomimetic exosome vesicles (apoA1-bExo) which were then incubated with cholesterol modified siRNA to generate apoA1-bExo containing siRNA (apoA1-bExo/siRNA). Thepreparedvesicleswere uniformandsphericalin size and could be loaded effectively with siRNA to protect from nuclease degradation. Compared with bExo/siRNA, apoA1-bExo/siRNA showed stronger tumor targeting, tissue permeability, intracellular accumulation efficiency and antitumor efficiency. A portion of apoA1-bExo/siRNA transport siRNA occurred through the endosome-Golgi-ER pathway similar to bExo/siRNA, but mostly occurred directly through selective uptake pathways mediated by the SR-B1 receptor. apoA1-bExo/siRNA successfully achieved silencing efficiency at the transcription and protein levels (96.78 % and 94.07 %, respectively) and reduced the secretion of ExoPD-L1 from HepG2 cells to 15.92 % of that in the PBS group, thus enhancing the killing activity of co-cultured T cells on HepG2 cells. In addition, relevant pharmacodynamic indices were positively correlated with delivery efficiency and the modification of apoA1 could significantly enhance the intracellular accumulation of siRNA, thus exhibiting stronger activity than bExo/siRNA. Moreover, in addition to curing mice of their implanted tumors, blocking ExoPD-L1 secretion in combination with αPD-1 promoted the infiltration of durable antitumor hCD8+ T cells and hCD45+ T cells into tumor in a immune system-tumor dual humanized mice.

Novel Avian Influenza Virus (H5N1) Clade 2.3.4.4b Reassortants in Migratory Birds, China.

Two novel reassortant highly pathogenic avian influenza viruses (H5N1) clade 2.3.4.4b.2 were identified in dead migratory birds in China in November 2021. The viruses probably evolved among wild birds through different flyways connecting Europe and Asia. Their low antigenic reaction to vaccine antiserum indicates high risks to poultry and to public health.

Implementation effect of a hierarchical pharmaceutical service pattern in patients with systemic lupus erythematosus.

OBJECTIVES: This study evaluated the effect of implementing a hierarchical pharmaceutical service pattern based on the knowledge-attitude-practice (KAP) intervention theory on patients with systemic lupus erythematosus. METHODS: Eligible patients were randomly divided into an intervention or control group. Pharmaceutical service classification criteria were formulated and used to provide patients with differing levels of pharmaceutical services. The classification scores and KAP levels of patients before and at various time points after the intervention were analyzed. The rates of acute attacks and adverse reactions, related clinical test indices, and disease activity were evaluated in both groups. RESULTS: After 9 months of intervention, the proportions of first- and second-level services in the intervention group declined by 14.43% and 3.94%, respectively, compared with the control group, and the rates of acute attacks and adverse reactions declined by 18.26% and 12.43%, respectively. The KAP level, clinical test indices, and disease activity were significantly different between the groups. CONCLUSION: Providing patients with systemic lupus erythematosus with pertinent hierarchical pharmaceutical services based on the KAP theory was instrumental in changing patients' behavior and contributed to facilitating disease self-management, thus improving the quality of pharmaceutical services.

Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chickens, a pet cat, and humans at a backyard farm in Guangxi, China.

During an investigation in October 2018, two people with diarrhoea, mild abdominal pain, and mild arthralgia symptoms in Guangxi, China, were identified as infected by H9N2 avian influenza virus (AIV). Four H9N2 AIVs were isolated from one of two patients, a pet cat, and a dead chicken (two respective isolates from its lung and kidney tissues) bred by the patients at a backyard farm. Epidemiological investigation indicated that the newly bought chicken died first, and clinical syndromes appeared subsequently in the two owners and one cat. Furthermore, the two individuals possessed high H9N2-specific hemagglutination inhibition and microneutralization antibodies. Shared nucleotide sequence identity (99.9% - 100%) for all genes was detected in the four H9N2 isolates, and hemagglutinin (HA) T138A located on the receptor binding domain (RBD), resulted from nucleotide polymorphisms that were exclusively found in the isolate from the female patient. Moreover, HA K137N on the RBD was found in isolates from these three host species. Importantly, these four H9N2 isolates presented an exclusive binding preference for the human-type receptor (α2-6-SA), and could replicate and cause pathological changes in mice. Phylogenetic analyses showed that these four isolates clustered together and belonged to clade C1.2, lineage Y280. In addition, H9N2 viruses of human origin are genetically divergent and interspersed with the widespread poultry-origin H9N2 AIVs. All these results indicate a high risk of H9N2 AIVs in public health, and effective prevention and control measures against H9N2 AIVs should be considered and performed for both animal and human health.

Co-existence and co-infection of influenza A viruses and coronaviruses: Public health challenges.

Since the 20th century, humans have lived through five pandemics caused by influenza A viruses (IAVs) (H1N1/1918, H2N2/1957, H3N2/1968, and H1N1/2009) and the coronavirus (CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IAVs and CoVs both have broad host ranges and share multiple hosts. Virus co-circulation and even co-infections facilitate genetic reassortment among IAVs and recombination among CoVs, further altering virus evolution dynamics and generating novel variants with increased cross-species transmission risk. Moreover, SARS-CoV-2 may maintain long-term circulation in humans as seasonal IAVs. Co-existence and co-infection of both viruses in humans could alter disease transmission patterns and aggravate disease burden. Herein, we demonstrate how virus-host ecology correlates with the co-existence and co-infection of IAVs and/or CoVs, further affecting virus evolution and disease dynamics and burden, calling for active virus surveillance and countermeasures for future public health challenges.

Re-emergence of H5N8 highly pathogenic avian influenza virus in wild birds, China.

In mid-November 2020, deaths of whooper swan were reported in the Yellow River Reservoir Area, China. In the present study, we describe the genetic characterizations and phylogenetic relationships of four clade 2.3.4.4b H5N8 highly avian influenza viruses (HPAIVs) identified from a sick whooper swan and environmental samples collected in the Yellow River Reservoir Area in late November 2020. They were closely related to recent H5Nx HPAIVs causing outbreaks in Eurasia in the 2020-2021 influenza season, suggesting these isolates might be imported into China via migratory birds. The newly identified H5N8 HPAIVs possessed Q226 and G228 (H3 numbering), indicating that they prefer to avian-like receptors. However, they had three mutations falling within known antigenic regions, including T144A in antigenic region A, T192I in antigenic region B, and N240D in antigenic region D. Our study highlights the risk of the rapid global spread of H5N8 HPAIVs and the necessity for continuous monitoring of avian influenza viruses in wild birds.

Distinctive polymer micelle designed for siRNA delivery and reversal of MDR1 gene-dependent multidrug resistance.

P-glycoprotein (P-gp) plays an importantrole in multidrug resistance (MDR), proved to be one of the major obstacles in cancer chemotherapy. Cationic polymers could specifically deliver siRNA to tumor cells and thus reverse MDR by the downregulation of P-gp. In this study, a triblock copolymer micelle was prepared based on the polymer of N-succinyl-chitosan-poly-l-lysine-palmitic acid (NSC-PLL-PA) to deliver siRNA-P-gp (siRNA-micelle) or doxorubicin (Dox-micelle). The resulting micelle exhibited an efficient binding ability for siRNA and high encapsulation efficiency for Dox, with an average particle size of ∼170 nm. siRNA-micelle and Dox-micellewere instable at low pH, thereby enhancing tumor accumulation and intracellular release of the encapsulated siRNA and Dox. siRNA-micelle micelles could enhance the knockdown efficacy of siRNA by improving the transfection efficiency, downregulating P-gp expression, and passing the drug efflux transporters, thereby improving the therapeutic effects of Dox-micelle. However, P-gp could transfer from HepG2/ADM to HepG2 cells independent of the expression of mdr1, and the acquired resistance could permit tumor cells to survive and develop intrinsic P-gp-mediated resistance, thereby limiting the desired efficiency of chemotherapeutics. This study demonstrated the effectiveness of siRNA-micelle for tumor-targeted delivery, MDR reversal, and provided an effective strategy for the treatment of cancers that develop MDR. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2093-2106, 2017.

Novel polymer micelle mediated co-delivery of doxorubicin and P-glycoprotein siRNA for reversal of multidrug resistance and synergistic tumor therapy.

Co-delivery of chemotherapeutics and siRNA with different mechanisms in a single system is a promising strategy for effective cancer therapy with synergistic effects. In this study, a triblock copolymer micelle was prepared based on the polymer of N-succinyl chitosan-poly-L-lysine-palmitic acid (NSC-PLL-PA) to co-deliver doxorubicin (Dox) and siRNA-P-glycoprotein (P-gp) (Dox-siRNA-micelle). Dox-siRNA-micelle was unstable in pH 5.3 medium than in pH 7.4 medium, which corresponded with the in vitro rapid release of Dox and siRNA in acidic environments. The antitumor efficacy of Dox-siRNA-micelle in vitro significantly increased, especially in HepG2/ADM cells, which was due to the downregulation of P-gp. Moreover, almost all the Dox-siRNA-micelles accumulated in the tumor region beyond 24 h post-injection, and the co-delivery system significantly inhibited tumor growth with synergistic effects in vivo. This study demonstrated the effectiveness of Dox-siRNA-micelles in tumor-targeting and MDR reversal, and provided a promising strategy to develop a co-delivery system with synergistic effects for combined cancer therapy.

Liposomes coated with N-trimethyl chitosan to improve the absorption of harmine in vivo and in vitro.

In this study, harmine liposomes (HM-lip) were prepared through the thin-film hydration-pH-gradient method and then coated with N-trimethyl chitosan (TMC). Particle size, zeta potential, entrapment efficiency, and in vitro release of HM-lip and TMC-coated harmine liposomes (TMC-HM-lip) were also determined. Sprague Dawley rats were further used to investigate the pharmacokinetics in vivo. Retention behavior in mouse gastrointestinal tract (GIT) was studied through high-performance liquid chromatography and near-infrared imaging. Degradation was further evaluated through incubation with Caco-2 cell homogenates, and a Caco-2 monolayer cell model was used to investigate the uptake and transport of drugs. HM-lip and TMC-HM-lip with particle size of 150-170 nm, an entrapment efficiency of about 81%, and a zeta potential of negative and positive, respectively, were prepared. The release of HM from HM-lip and TMC-HM-lip was slower than that from HM solution and was sensitive to pH. TMC-HM-lip exhibited higher oral bioavailability and had prolonged retention time in GIT. HM-lip and TMC-HM-lip could also protect HM against degradation in Caco-2 cell homogenates. The uptake amount of TMC-HM-lip was higher than that of HM and HM-lip. TMC-HM-lip further demonstrated higher apparent permeability coefficient (P(app)) from the apical to the basolateral side than HM and HM-lip because of its higher uptake and capability to open tight junctions in the cell monolayers. TMC-HM-lip can prolong the retention time in the GIT, protect HM against enzyme degradation, and improve transport across Caco-2 cell monolayers, thus enhancing the oral bioavailability of HM.

CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells.

An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice.

Systemic delivery of micelles loading with paclitaxel using N-succinyl-palmitoyl-chitosan decorated with cRGDyK peptide to inhibit non-small-cell lung cancer.

This study aimed to prepare efficient cRGDyK peptide-decorated micelles for the targeted therapy of non-small-cell lung cancer (NSCLC). An amphiphilic copolymer N-succinyl-palmitoyl-chitosan (SPCS) was synthesized and characterized. cRGDyK peptide is a ligand that can target tumors via specific binding integrin receptor overexpressed on tumor neovascularization and cells. cRGDyK-functionalized SPCS micelles loaded with paclitaxel (PTX/cRGDyK-SPCS) were prepared by film dispersion method and then characterized according to morphology, size, and zeta potential. PTX/cRGDyK-SPCS micelles presented pH-triggered drug release behavior under acidic conditions. The accumulation of micelles detected by laser confocal fluorescence microscopy and flow cytometry showed that cRGDyK-SPCS micelles were easily taken up by A549 cells marked with the luciferase gene (luc-A549). Meanwhile, co-localization of the micelles and lysosomes was recorded dynamically using a live cell station. MTT assays and cell apoptosis studies revealed that cell viability was significantly inhibited by PTX/cRGDyK-SPCS micelles. More importantly, in vivo animal studies showed that cRGDyK-SPCS micelles mainly accumulated in the orthotopic tumor site. PTX/cRGDyK-SPCS micelles exhibited better anti-tumor activity in subcutaneous and orthotopic lung tumors compared with PTX/SPCS micelles and Taxol(®). These results suggested that PTX/cRGDyK-SPCS micelles had better cancer targeting capacity and superior anti-tumor efficacy. Thus, these micelles have great potential as novel carriers in delivering anti-tumor drugs.

N-Succinyl-chitosan nanoparticles coupled with low-density lipoprotein for targeted osthole-loaded delivery to low-density lipoprotein receptor-rich tumors.

N-Succinyl-chitosan (NSC) was synthesized and NSC nanoparticles (NPs) with loaded osthole (Ost) (Ost/NSC-NPs) were prepared by emulsion solvent diffusion. Subsequently, low-density lipoprotein (LDL)-mediated NSC-NPs with loaded Ost (Ost/LDL-NSC-NPs) were obtained by coupling LDL with Ost/NSC-NPs through amide linkage. The average particle size of Ost/NSC-NPs was approximately 145 nm, the entrapment efficiency was 78.28%±2.06%, and the drug-loading amount was 18.09%±0.17%. The release of Ost from Ost/NSC-NPs in vitro showed a more evident sustained effect than the native material. The half maximal inhibitory concentration of Ost/LDL-NSC-NPs was only 16.23% that of the free Ost at 24 hours in HepG2 cells. Ost inhibited HepG2 cell proliferation by arresting cells in the synthesis phase of the cell cycle and by triggering apoptosis. Cellular uptake and subcellular localization in vitro and near-infrared fluorescence real-time imaging in vivo showed that Ost/LDL-NSC-NPs had high targeting efficacy. Therefore, LDL-NSC-NPs are a promising system for targeted Ost delivery to liver tumor.

Low-density lipoprotein-coupled N-succinyl chitosan nanoparticles co-delivering siRNA and doxorubicin for hepatocyte-targeted therapy.

Developing safe and effective carriers of small interference RNA (siRNA) is a significant demand for the systemic delivery of siRNA. In this study, low-density lipoprotein (LDL) was isolated from human plasma and loaded with cholesterol-conjugated siRNA to silence the multidrug resistant gene of tumors. Chol-siRNA/LDL-coupled N-succinyl chitosan nanoparticles loaded with doxorubicin (Dox-siRNA/LDL-SCS-NPs) were then prepared and characterised. The Dox-siRNA/LDL-SCS-NPs had average particle size of 206.4 ± 9.2 nm, entrapment efficiency of 71.06% ± 1.42%, and drug-loading amount of 12.35% ± 0.87%. In vitro antitumor activity revealed that cell growth was significantly inhibited. The accumulation of Dox by fluorescence microscopy and flow cytometry showed that LDL-coupled nanoparticles were more easily taken up than Dox-SCS-NPs. Results of confocal microscopy and reverse transcription-PCR revealed the highly efficient uptake of siRNA and the decrease in mdr1 mRNA expression. LDL-coupled nanoparticles protected siRNA from macrophage phagocytosis by dynamic observation using live cell station. In vivo tumor-targeting suggested that Cy7-labelled Dox-LDL-SCS-NPs were markedly accumulated in an analyzed in situ liver tumor model. Results indicated that LDL-SCS-NPs were effective tumor-targeting vectors and that the preparation form may provide a new strategy for co-delivering siRNA and antitumor drugs.

Novel self-assembled micelles based on palmitoyl-trimethyl-chitosan for efficient delivery of harmine to liver cancer.

BACKGROUND: Polymeric micelles is a safe and effective delivery system, which belong to the targeted delivery system (TDS). An anticancer drug, harmine(HM) is a hydrophobic drug with much adverse effects when used for treatment of liver cancer. Chitosan (CS) is a polysaccharide and can be modified to be an amphiphilic polmer which could self-assemble into micelles and be applied for delivery of hydrophobic drugs. OBJECTIVES: To synthesize three kinds of novel biodegradable polymers, designated as palmitoyl-trimethyl-CS (TPCS)1, TPCS2 and Lac-TPCS2, and investigate their efficiency and mechanism of delivery HM to liver tumors in vitro and in viro. RESULTS: The self-assembled micelles presented satisfactory particle size (∼ 200 nm) and drug release characteristics in vitro. It's proved that Lac-TPCS2/HM may enter HepG2 cell through endocytosis. Antitumor experiments in vivo revealed that Lac-TPCS2/HM could significantly inhibit tumor growth and extend the lifetime of mice bearing H22 tumors after intravenous administration. Subsequently in vivo near-infrared fluorescence imaging results demonstrated a satisfactory liver tumor-targeting effect of Lac-TPCS2/HM. CONCLUSION: Three novel polymers hold great potential in the development of nanomedicine for treatment of liver tumors, in particular Lac-TPCS2 exhibits the greatest antitumor potential through active target effect.

Synthesis and characterization of low-toxicity N-caprinoyl-N-trimethyl chitosan as self-assembled micelles carriers for osthole.

Novel amphiphilic chitosan derivatives (N-caprinoyl-N-trimethyl chitosan [CA-TMC]) were synthesized by grafting the hydrophobic moiety caprinoyl (CA) and hydrophilic moiety trimethyl chitosan to prepare carriers with good compatibility for poorly soluble drugs. Based on self-assembly, CA-TMC can form micelles with sizes ranging from 136 nm to 212 nm. The critical aggregation concentration increased from 0.6 mg • L(-1) to 88 mg • L(-1) with decrease in the degree of CA substitution. Osthole (OST) could be easily encapsulated into the CA-TMC micelles. The highest entrapment efficiency and drug loading of OST-loaded CA-TMC micelles(OST/CA-TMC) were 79.1% and 19.1%, respectively. The antitumor efficacy results show that OST/CA-TMC micelles have significant antitumor activity on Hela and MCF-7 cells, with a 50% of cell growth inhibition (IC50) of 35.8 and 46.7 µg. mL(-1), respectively. Cell apoptosis was the main effect on cell death of Hela and MCF-7 cells after OST administration. The blank micelles did not affect apoptosis or cell death of Hela and MCF-7 cells. The fluorescence imaging results indicated that OST/CA-TMC micelles could be easily uptaken by Hela and MCF-7 cells and could localize in the cell nuclei. These findings suggest that CA-TMC micelles are promising carriers for OST delivery in cancer therapy.

Application of the central composite design to optimize the preparation of novel micelles of harmine.

Lactose-palmitoyl-trimethyl-chitosan (Lac-TPCS), a novel amphipathic self-assembled polymer, was synthesized for administration of insoluble drugs to reduce their adverse effects. The central composite design was used to study the preparation technique of harmine (HM)-loaded self-assembled micelles based on Lac-TPCS (Lac-TPCS/HM). Three preparation methods and single factors were screened, including solvent type, HM amount, hydration volume, and temperature. The optimal preparation technique was identified after investigating the influence of two independent factors, namely, HM amount and hydration volume, on four indexes, ie, encapsulation efficiency (EE), drug-loading amount (LD), particle size, and polydispersity index (PDI). Analysis of variance showed a high coefficient of determination of 0.916 to 0.994, thus ensuring a satisfactory adjustment of the predicted prescription. The maximum predicted values of the optimal prescription were 91.62%, 14.20%, 183.3 nm, and 0.214 for EE, LD, size, and PDI, respectively, when HM amount was 1.8 mg and hydration volume was 9.6 mL. HM-loaded micelles were successfully characterized by Fourier-transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, and a fluorescence-quenching experiment. Sustained release of Lac-TPCS/HM reached 65.3% in 72 hours at pH 7.4, while free HM released about 99.7% under the same conditions.