The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Identification of Hemagglutinin Mutations Caused by Neuraminidase Antibody Pressure.

The balance in the functions of hemagglutinin (HA) and neuraminidase (NA) plays an important role in influenza virus genesis. However, whether and how N2 neuraminidase-specific antibodies may affect the attributes of HA remains to be investigated. In this study, we examined the presence of amino acid mutations in the HA of mutants selected by incubation with N2-specific monoclonal antibodies (MAbs) and compared the HA properties to those of the wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus. The higher NA inhibition (NI) ability of N2-specific MAbs was found to result in greater proportions of mutations in the HA head. The HA mutations affected the thermal stability, switched the binding preferences from α2,6-linked sialic acid receptor to α2,3-linked sialic acid receptor, and promoted viral growth in mouse lungs. These mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition (HI) titers. The evolutionary analysis also proved that some HA mutations were highly correlated with NA antibody pressure. Our data demonstrate that HA mutations caused by NA-specific antibodies affect HA properties and may contribute to HA evolution. IMPORTANCE HA binds with the sialic acid receptor on the host cell and initiates the infection mode of influenza virus. NA cleaves the connection between receptor and HA of newborn virus at the end of viral production. The HA-NA functional balance is crucial for viral production and interspecies transmission. Here, we identified mutations in the HA head of H9N2 virus caused by NA antibody pressure. These HA mutations changed the thermal stability and switched the receptor-binding preference of the mutant virus. The HI results indicated that these mutations resulted in significant antigenic drift in mutant HA. The evolutionary analysis also shows that some mutations in HA of H9N2 virus may be caused by NA antibody pressure and may correlate with the increase in H9N2 infections in humans. Our results provide new evidence for HA-NA balance and an effect of NA antibody pressure on HA evolution.

Radiomic features as a risk factor for early postoperative seizure in patients with meningioma.

PURPOSE: This study aim to identify the clinical risk factors of and to develop a radiomics-based predictive model for early postoperative seizure. METHODS: We retrospectively assessed 322 operative patients with meningioma who met the inclusion criteria from January 2014 to December 2016 at The First Affiliated Hospital of Wenzhou Medical University. Univariate and multivariate analyses were performed to determine the predictive value of clinical variables. Magnetic resonance imaging (MRI) was performed to obtain the radiomic score (Rscore) for early postoperative seizure. Radiological features were evaluated using the AK software. The minimal redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) methods were used to assess for radiomic features, and the Rscore was obtained based on radiomic characteristics using a specific formula. RESULTS: In total, 260 patients who met the inclusion criteria were finally enrolled in this study. Among them, 20 experienced early postoperative seizure. Logistic regression analysis showed that Rscore was associated with a significantly high risk of seizure (p<0.000). Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve of the Rscore was 0.92 (95% confidence interval: 0.853-0.987). The model had a high accuracy for predicting early postoperative seizure. CONCLUSIONS: The Rscore was found to be associated with a high risk of early postoperative seizures. Thus, a higher Rscore (>-1.644) can identify high-risk patients requiring intensive care.

A Cross-Reactive Monoclonal Antibody Against Neuraminidases of Both H9N2 and H3N2 Influenza Viruses Shows Protection in Mice Challenging Models.

Neuraminidases (NAs) of H9N2 avian influenza virus (AIV) and H3N2 human seasonal influenza virus (HSIV) share similar antigenic structures. However, there are few reports on epitopes shared by these two NAs. We previously reported a monoclonal antibody (mAb) 1G8 against the NA of H9N2 AIV with neuraminidase inhibition (NI) ability. In this study, 1G8 was shown to cross-react with and inhibit the NA of H3N2 HSIV. In a passive transfer experiment, 1G8 provided protection to mice challenged with rescued H1N2 viruses carrying H9N2 NA or H3N2 NA. Mutation at amino acid position 199 was also selected and proved to be crucial for H3N2 HSIV to escape from mAb 1G8. Moreover, we found that residue 199 contributed to inducing broad protective antibodies without the influence of the N-linked glycosylation at amino acid position 200 in NAs. Residues as residue 199, which are not shielded by glycosylation modification, would form ideal epitopes for developing universal vaccine and protective antibodies.

Tumor stem-like cells isolated from MMQ cells resist to dopamine agonist treatment.

Although tumor stem-like cells (TSLCs) have been studied in a range of malignant tumors, evidence for the presence of these cells in pituitary adenomas needs further exploration. Here, we identified a small subset of sphere-forming cells possess tumor stem-like cell properties in rat prolactinoma MMQ cells, which resist to dopamine agonist treatment. Comparing to MMQ cells, sphere-forming cells showed higher cell viability after dopamine agonist (DA) treatment. Furthermore, the cells showed lower expression of prolactin (PRL) and dopamine 2 receptor (D2R). On the contrary, the daughter tumor cells differentiated from these cells restored the sensitivity to DA and showed high expression of PRL and D2R. The lower D2R expression and DA resistance might be due to DNA hypermethylation of D2R promoter. Our study demonstrates that the sphere-forming cells isolated from MMQ cells possess the trait of TSLCs and resist to DA treatment, which offers the opportunity to further investigate the mechanisms underlying tumor recurrence based on TSLCs.

Peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine.

BACKGROUND: Infectious bronchitis virus (IBV), a coronavirus, is one of the most important poultry pathogens worldwide due to its multiple serotypes and poor cross-protection. Vaccination plays a vital role in controlling the disease. The efficacy of vaccination in chicken flocks can be evaluated by detecting neutralizing antibodies with the neutralization test. However there are no simple and rapid methods for detecting the neutralizing antibodies. RESULTS: In this study, a peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine was developed. The pELISA could indirect evaluate neutralizing antibody titers against different types of IBV in all tested sera. The titers measured with the pELISA had a coefficient of 0.83 for neutralizing antibody titers. CONCLUSIONS: The pELISA could detect antibodies against different types of IBV in all tested sera. The pELISA has the potential to evaluate samples for IBV-specific neutralizing antibodies and surveillance the infection of IBV.

Identification of key residues involved in the neuraminidase antigenic variation of H9N2 influenza virus.

Influenza A H9N2 virus causes economic loss to the poultry industry and has likely contributed to the genesis of H5N1 and H7N9 viruses. The neuraminidase (NA) of H9N2 virus, like haemagglutinin, is under antibody selective pressure and may undergo antigenic change; however, its antigenic structure remains to be elucidated. In this study, we used monoclonal antibodies (mAbs) to probe the H9N2 viral NA residues that are key for antibody binding/inhibition. These mAbs fell into three groups based on their binding/inhibition of the NA of H9N2 viruses isolated during 1999-2019: group I only bounded the NA of the early 2000 H9N2 viruses but possessed no neutralizing ability, group II bounded and inhibited the NA of H9N2 viruses isolated before 2012, and group III reacted with most or all tested H9N2 viruses. We showed that NA residue 356 is key for the recognition by group I mAbs, residues 344, 368, 369, and 400 are key for the binding/inhibition of NA by group II antibodies, whereas residues 248, 253, and the 125/296 combination are key for neutralizing antibodies in group III. Our findings highlighted NA antigenic change of the circulating H9N2 viruses, and provided data for a more complete picture of the antigenic structure of H9N2 viral NA.

MicroRNA-93 mediates cabergoline-resistance by targeting ATG7 in prolactinoma.

To date, the management of dopamine agonist (DA)-resistant prolactinomas remains a major clinical problem. Previously, we determined that miRNA-93 expression increases in DA-resistant prolactinomas; however, the role of miRNA-93 in the DA resistance remains largely unexplored. Hence, this study aimed to investigate the susceptibility of tumor cells to cabergoline (CAB) and the autophagy changes in MMQ and GH3 cells after miRNA-93 overexpression or inhibition. We used bioinformatics to identify the potential target of miRNA-93. Subsequently, we analyzed the correlation between miRNA-93 and autophagy-related 7 (ATG7) using protein expression analysis and luciferase assays. Furthermore, the change in the effect of miRNA-93 was measured after ATG7 overexpression. miRNA-93 expression was elevated in DA-resistant prolactinomas, whereas the expression of its identified target, ATG7, was downregulated. miRNA-93 overexpression suppressed the cytotoxic effect of CAB in MMQ and GH3 cells. In contrast, miRNA-93 downregulation enhanced CAB efficiency and promoted cell autophagy, eventually resulting in apoptosis. These results were further confirmed in vivo xenograft models in nude mice. ATG7 overexpression could reverse the inhibitory effect of miRNA-93 on CAB treatment. Taken together, our results suggest that miRNA-93 mediates CAB resistance via autophagy downregulation by targeting ATG7 and serves as a promising therapeutic target for prolactinoma.

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells.

BACKGROUND: Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest. METHODS: The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student's t-test was used to compare differences between treated groups and their controls. RESULTS: We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice. COCLUSIONS: In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.

Dopamine receptor D2S gene transfer improves the sensitivity of GH3 rat pituitary adenoma cells to bromocriptine.

Bromocriptine, a dopamine agonist (DA), has been used in the treatment of prolactinomas. Recent studies have indicated that dopamine 2 receptor short isoform (D2S) may play an important role in suppressing PRL synthesis and prolactinoma cell growth under DA treatment. In the current study, we investigated the role of D2S in the therapeutic action of bromocriptine in GH3 using both in vitro and in vivo approaches. Infection of adenovirus-D2S increased D2S expression in GH3 cells (P<0.05). D2S expression significantly decreased the GH3 cell viability subjected to bromocriptine treatment in vitro (P<0.05). In nude mice, adenovirus-D2S transfection sensitized GH3 xenograft to bromocriptine treatment evidenced by the significant inhibition of D2S expressed tumor growth as compared with vector control. Furthermore, decrease of Bcl-2 expression, increase of Bax, and active Caspase-3 were found in D2S expressed GH3 xenograft subjected to bromocriptine treatment. In summary, our study indicates that D2S expression plays a critical role in the therapeutic action of bromocriptine in pituitary adenomas and that adenovirus-mediated D2S gene transfer combined with bromocriptine may provide a novel treatment for DA-resistant prolactinomas.

Differential effects of nerve growth factor on expression of dopamine 2 receptor subtypes in GH3 rat pituitary tumor cells.

Nerve growth factor (NGF) can increase expression of dopamine 2 receptors (D2R) in GH3 rat pituitary tumor cells (GH3 cells). As D2R exists in long (D2L) and short (D2S) isoforms, effects of NGF on D2R subtypes have not been accurately evaluated. In this study, we compared mRNA levels of D2R subtypes in GH3 cells treated with or without NGF with real-time RT-PCR. In addition, we also evaluated the relationship between GH3 cell growth after bromocriptine treatment and mRNA levels of D2R subtypes. We found that D2R total, D2L, and D2S mRNA in GH3 cells were significantly increased after NGF treatment, compared with the vehicle group. Moreover, NGF increased the ratio of D2S to D2L. GH3 cell survival rate after bromocriptine treatment was negatively correlated with D2R total mRNA, and D2S may be more potent than D2L in inhibiting cell growth. Cell apoptosis rate was highly elevated in GH3 cells treated with NGF and bromocriptine, compared with the control group or the group treated with NGF or bromocriptine alone. Our data provide preliminary evidence that the effect of NGF was more prominent on expression of D2S than D2L, in addition, D2S might have a greater impact suppressing GH3 cells growth than D2L.

microRNA expression profile and differentially-expressed genes in prolactinomas following bromocriptine treatment.

Little is known about the function of microRNAs in prolactinomas treated with bromocriptine. The aim of the study was to explore the microRNAs associated with bromocriptine-treated prolactinomas. Six prolactinoma samples were selected according to whether they received bromocriptine treatment or not before microsurgery, and microRNA expression profiles of bromocriptine-treated and untreated prolactinomas were screened by the miRCURY LNA Array. The differentially expressed microRNAs in microarrays were further validated by stem-loop real-time PCR and subjected to gene ontology analysis and KEGG pathway analysis. In addition, related genes of microRNAs were analyzed by qRT-PCR in 15 prolactinoma samples. The initial analysis by microarrays generated a list of 80 upregulated microRNAs and 71 downregulated microRNAs in treated prolactinomas compared to untreated prolactinomas. miR-206, miR-516b and miR-550 were confirmed to be significantly upregulated, while miR-671-5p was confirmed to be significantly downregulated in treated prolactinomas by qRT-PCR. microRNA-mRNA network analysis integrating GO and KEGG pathway annotation displayed some critical factors. Platelet-derived growth factor α polypeptide (PDGFA) and bone morphogenetic protein 4 (BMP4), were verified to be differentially expressed between the two groups. PDGFA was significantly upregulated in treated prolactinomas, while BMP4 was significantly downregulated in treated prolactinomas. Our study reveals differential expression of microRNAs in prolactinoma after pharmacotherapy. Specific microRNAs may be involved in the inhibition or promotion of prolactinoma tumor growth impacted by bromocriptine pharmacotherapy. PDGFA and BMP4 may be involved in the pharmacotherapy mechanism of prolactinoma.

[Inhibitory effect of adenovirus-mediated D2S gene plus bromocriptine therapy on primary cultures of human non-functioning pituitary adenoma cells].

OBJECTIVE: To explore the inhibitory effect of non-functioning pituitary adenoma (NFPA) cells after a combined treatment of adenovirus mediated D2S gene and bromocriptine in vitro. METHODS: Adenovirus containing dopamine 2 receptor short isoform (D2S) gene was used to infect NFPA cells. The transfection of D2S gene into NFPA cells was confirmed by immunofluorescence. And cell apoptosis of infected cells treated by bromocriptine was evaluated with CCK-8 assay in vitro. RESULTS: When D2S gene transfection and bromocriptine was used in combination, the survival rate of NFPA cells significantly decreased (40 ± 5)% versus the control group (97 ± 5)% and the pAd-EGFP transfection combined bromocriptine treatment group (90 ± 9)% (P < 0.05). CONCLUSION: The combined treatment of adenovirus-mediated D2S gene and bromocriptine can effectively induce the apoptosis of NFPA cells on primary culture and increase the sensitivity of NFPA to dopamine agonist.

Expression of dopamine 2 receptor subtype mRNA in clinically nonfunctioning pituitary adenomas.

Dopamine receptor agonists (DAs) can reduce hormone release and tumor mass in the majority of prolactinomas, whereas such effects are controversial in clinically nonfunctioning pituitary adenomas (NFPAs). Whether expression of dopamine 2 receptor (D2R) is different in subgroups of NFPAs has not been fully elucidated. We assessed and compared D2R subtype (long: D2L and short: D2S) mRNA levels in subgroups of NFPAs by real-time reverse transcriptase polymerase chain reaction (RT-PCR). For both D2L and D2S mRNA, there were no significant differences among them. Only 21.6% of NFPAs showed relatively high D2R mRNA levels; furthermore, histopathological subtypes of those cases with relatively high D2R expression were gonadotropinomas and null-cell adenomas. These data suggest that DAs are effective only for a small proportion of NFPAs, and relatively high D2R expression may more possibly happen to a subset of gonadotropinomas and null-cell adenomas.

[Natural history study of postoperative residual non-functioning pituitary adenomas].

OBJECTIVE: To observe the postoperative residual non-functioning pituitary adenomas (PR-NFPAs) without postoperative radiotherapy and to analyze the natural history of PR-NFPAs' growth in order to provide a basis for selecting appropriate strategies of clinical treatment. METHODS: We evaluated the natural history of 20 patients with PR-NFPAs who did not receive postoperative radiotherapy and drug therapy. Through MRI images, the residual tumor volumes of those patients were serially measured. We further calculated the monthly growth rate and the tumor volume doubling time (TVDT) and analyzed the correlations between the patient age, gender, volume of residual tumor, cavernous sinus (CS) invasion and TVDT. RESULTS: All patients received observation alone. Among which, 17 adenomas increased in volume and 3 remained unchanged during a follow-up period of 7 months to 17 years (mean 3.90 yr). The mean patient age was 41.8 years. As to 17 patients with tumor regrowth, the tumor volume at the beginning of MRI observation period was 4.73 cm(3) and tumor volume at the last MRI observation was 16.98 cm(3). During the mean 4-year follow-up period, the average monthly growth rate of PR-NFPAs was 7.87% and the mean TVDT was 724 days. Such factors as patient age, gender, volume of residual tumor and CS invasion did not affect the TVDT of PR-NFPAs. CONCLUSION: The tumor growth rate of PR-NFPAs is not significantly correlated with the patient gender, age, volume of residual tumor and CS invasion. In conjunctions with the volume of PR-NFPAs and the distance between residual adenoma and optic chiasm, we should take the TVDT into consideration and determine the appropriate and safe follow-up period.

Killing effect of interleukin-13 receptor alpha 2 (IL-13Ralpha2) sensitized DC-CTL cells on human glioblastoma U251 cells.

Recent studies show that IL-13Ralpha2, a brain tumor-associated antigen for IL-13, may play a role in immunotherapy for glioblastoma. Thus, we stimulated the lymphocyte by monocyte-derived dendritic cells (DCs). The DCs were pulsed with IL-13Ralpha2 in vitro and then co-cultured with lymphocytes. After inducing cytotoxic T cells (CTLs) and co-culturing with U251 cells for 24 h in 96 wells, Cell Count Kit-8 (CCK-8) was added to every well equally. The optical density (OD) value was detected and recorded after 2 h. The DCs efficiently presented the antigen to the CTLs, resulting in CTLs activation and proliferation. The induced CTLs showed specific cytotoxic against U251 cells (P<0.01). The results demonstrated that IL-13Ralpha2 induced CTLs could kill glioma U251 in vitro, which suggests that IL-13 Ralpha2 might have such an impact in vivo and thus recombinant IL-13Ra2 protein might be used as an anti-tumor vaccine, providing a promising new strategy for the treatment of brain malignant gliomas.