Pilot-scale production of a highly efficacious and stable monoglycosylated influenza split virus vaccine.

The yearly epidemics and unpredictable outbreaks of influenza have raisedserious concernsglobally and led to prioritizing the development of an effective vaccine toprotectagainst newly emerging variants. Previously, we demonstrated that monoglycosylated influenza virus vaccines derived from A/California/7/2009 or an updated A/Brisbane/02/2018 (IVR-190) vaccine strain recommended by WHO are superior to fully glycosylated vaccines and could broadly protect against past and new coming H1N1 variants. However, whether such a monoglycosylated virus vaccine can be mass-produced to meet clinical demands and stable enough to provide consistent efficacy against H1N1 viruses remains unclear. Herein, we developed a platform for the pilot-scale production of the monoglycosylated split virus vaccine from the IVR-190 strain (IVR-190mg) with a robust and cost-effective manufacturing process. The critical parameters of inoculum dose, concentration of kifunensine, and optimized Endo H treatment process were comprehensively investigated. Several aims for preclinical studies of IVR-190mg were achieved, including [i] the execution of three engineering batch runs to validate lot-to-lot consistency, [ii] the establishment of IVR-190mg specifications to meet the acceptance criteria of a conventional influenza vaccine, [iii] an investigation of the stability profile of IVR-190mg, and completion of a safety evaluation by conducting an animal toxicology study. The toxicology study under GLP guidance found no systemic toxicity after rabbits were vaccinated with IVR-190mg. The serological data showed that IVR-190mg is highly immunogenic and effective in inducing a cross-strain protective level of antibody immune responses, including hemagglutination-inhibition titers, viral neutralization activity, and broad HA- and NA-inhibiting antibody titers against past and new H1N1 viruses. In conclusion, this study provides efficacy and safety profiles of IVR-190mg for further clinical study and shows that this vaccine without a glycan shield has great potential to be safe and protective against H1N1 variants.

Monoglycosylated SARS-CoV-2 receptor binding domain fused with HAstem-scaffolded protein vaccine confers broad protective immunity against SARS-CoV-2 and influenza viruses.

The SARS-CoV-2 and influenza pandemics have posed a devastating threat to global public health. The best strategy for preventing the further spread of these respiratory viruses worldwide is to administer a vaccine capable of targeting both viruses. Here, we show that a novel monoglycosylated vaccine designed based on the influenza virus HAstem conserved domain fused with the SARS-CoV-2 spike-RBD domain (HSSRmg) can present proper antigenicity that elicits sufficient neutralization efficacy against various SARS-CoV-2 variants while simultaneously providing broad protection against H1N1 viruses in mice. Compared with the fully glycosylated HSSR (HSSRfg), HSSRmg induced higher ELISA titers targeting HAstem and spike-RBD and exhibited significantly enhanced neutralization activity against the Wuhan pseudovirus. The enhanced immune responses raised by JR300-adjuvanted HSSRmg compared to HSSRmg alone include more anti-HAstem and anti-spike-RBD antibodies that provide cross-protection against H1N1 challenges and cross-neutralization of SARS-CoV-2 pseudoviruses. Furthermore, the enhanced immune response raised by JR300-adjuvanted-HSSRmg skews toward a more balanced Th1/Th2 response than that raised by HSSRmg alone. Notably, HSSRmg elicited more plasma B cells and memory B cells, and higher IL-4 and IFN-γ cytokine immune responses than spike (S-2P) in mice with preexisting influenza-specific immunity, suggesting that B-cell activation most likely occurs through CD4+ T-cell stimulation. This study demonstrated that HSSRmg produced using a monoglycosylation process and combined with the JR300 adjuvant elicits superior cross-strain immune responses against SARS-CoV-2 and influenza viruses in mice compared with S-2P. JR300-adjuvanted HSSRmg has great potential as a coronavirus-influenza vaccine that provides dual protection against SARS-CoV-2 and influenza infections.

Pilot-scale production of inactivated monoglycosylated split H1N1 influenza virus vaccine provides cross-strain protection against influenza viruses.

Influenza epidemics and pandemics caused by newly emerging virus strains highlight an urgent need to develop a universal vaccine against viruses. Previously, a monoglycosylated X-181mg vaccine demonstrated that the HA possessing a single N-acetylglucosamine at each N-glycosylation site is superior to confer broader protection in mice than conventional vaccines. However, the greatest challenge in conducting clinical trials is the need to develop robust manufacturing processes capable of producing vaccines at the pilot scale with the desired stability, potency, and efficacy. Whether the monoglycosylated virus vaccine platform can be applied to the new vaccine strain in a timely manner and whether the mass-produced vaccine has the proper immunogenicity to induce cross-protective immunity remains unclear. Here, we show that a pilot-scale manufacturing process produced a monoglycosylated A/Brisbane/02/2018(H1N1) virus vaccine (IVR-190mg) with a single glycan at each glycosylation site of HA and NA. Compared with the fully glycosylated virus vaccine (IVR-190fg), the IVR-190mg provided broader cross-protection in mice against a wide range of H1N1 variants. The enhanced antibody responses induced by IVR-190mg immunization include higher hemagglutination-inhibition titers, higher neutralization activity, more anti-HA head domain, more anti-HA stem antibodies, higher neuraminidase activity inhibition titers, and notably, higher antibody-dependent cellular cytotoxicity. Additionally, the IVR-190mg also induced a more balanced Th1/Th2 response and elicited broader splenic CD4+ and CD8+ T-cell responses than IVR-190fg. This study demonstrated that IVR-190mg produced using a pilot-scale manufacturing process elicits comprehensive cross-strain immune responses that have great potential to substantially mitigate the need for yearly reformulation of strain-specific inactivated vaccines.

The small molecule chemical compound cinobufotalin attenuates resistance to DDP by inducing ENKUR expression to suppress MYH9-mediated c-Myc deubiquitination in lung adenocarcinoma.

The small molecule chemical compound cinobufotalin (CB) is reported to be a potential antitumour drug that increases cisplatin (DDP) sensitivity in nasopharyngeal carcinoma. In this study, we first found that CB decreased DDP resistance, migration and invasion in lung adenocarcinoma (LUAD). Mechanistic studies showed that CB induced ENKUR expression by suppressing PI3K/AKT signalling to downregulate c-Jun, a negative transcription factor of ENKUR. Furthermore, ENKUR was shown to function as a tumour suppressor by binding to ß-catenin to decrease c-Jun expression, thus suppressing MYH9 transcription. Interestingly, MYH9 is a binding protein of ENKUR. The Enkurin domain of ENKUR binds to MYH9, and the Myosin_tail of MYH9 binds to ENKUR. Downregulation of MYH9 reduced the recruitment of the deubiquitinase USP7, leading to increased c-Myc ubiquitination and degradation, decreased c-Myc nuclear translocation, and inactivation of epithelial-mesenchymal transition (EMT) signalling, thus attenuating DDP resistance. Our data demonstrated that CB is a promising antitumour drug and may be a candidate chemotherapeutic drug for LUAD patients.

A Multidisciplinary Team Approach to the Treatment of Liver Cirrhosis.

OBJECTIVE: To explore the feasibility of treating cirrhosis using a multidisciplinary team approach (MDT) and to pinpoint the key factors influencing its implementation. METHODS: The data of 307 patients with decompensated cirrhosis were studied retrospectively. Patients who received more than two treatment measures were assigned to the MDT group (n=228), and patients who received symptomatic medical drug treatment only were assigned to the traditional treatment group (n=79). The follow-up period ranged from 4 to 10 years, and the average follow-up period was 5.7 years. The results of the biochemical tests for hepatitis B virus deoxyribonucleic acid, hepatitis C virus ribonucleic acid, and autoantibodies to liver disease were analyzed. RESULTS: The differences in gender and Child-Pugh grade of liver function between the two groups were not statistically significant. The MDT group had obvious advantages over the traditional treatment group in occupational composition, etiology composition, 5-year survival rate and annual hospitalization times. The leading causes of death in the MDT group, in descending order, were liver cancer, infection, mesenteric thrombosis, and non-hepatic disease, and, in the medical treatment group, they were liver failure, gastrointestinal bleeding, infection, and liver cancer. There was a significant statistical difference between the two groups (p < 0.05). In the multidisciplinary treatment, etiological treatment was the most widely used treatment, accounting for 79.8%, followed by endoscopic treatment (33.3%), peritoneal drainage and ascites reinfusion (25%), splenectomy combined with devascularization (11.4%) and stem cell transplantation and liver transplantation (1.8%). CONCLUSION: An MDT can improve the efficacy and prognosis of patients with cirrhosis and improve patient compliance. After multi-disciplinary intervention, the mortality spectrum of long-term survival patients with cirrhosis changes, and the mortality rate of liver cancer and non-liver disease increases.

Highly sensitive nucleic acid detection and transaminase level in treatment decisions for Chinese patients with cirrhosis caused by hepatitis.

OBJECTIVE: To explore the usefulness of highly sensitive nucleic acid detection for assisting with the accurate antiviral treatment of patients with cirrhosis that was caused by hepatitis. METHODS: There were 377 patients with hepatitis B with cirrhosis and 119 patients with hepatitis C with cirrhosis, either as hospitalized patients and outpatients, who were enrolled into the study. Among them, 299 were men and 197 were women between 23 and 82 years of age. All patients were examined using a domestic HBV DNA/HCV RNA test, which was negative in 162 cirrhosis with hepatitis B and 54 cirrhosis with hepatitis C patients (HBV DNA/HCV RNA <500 IU/mL). Prediction and analysis of the HBV DNA load using alanine aminotransferase (ALT) level was based on receiver operating characteristics (ROC) curve analysis. RESULTS: For patients with hepatitis C with cirrhosis, after the antiviral therapy, ALT, HCV RNA, and Child-Pugh grade were significantly improved compared with before treatment. ROC analysis results showed that an ALT level of 29 IU/mL was the most sensitive cutoff value to judge a positive HBV DNA load (sensitivity 1.0, specificity 0.237, Youden index 0.763). CONCLUSION: Precise detection for patients with cirrhosis caused by hepatitis is required for accurate therapy.

Think twice before stent insertion for renal artery aneurysm with elusive etiology: a case report.

BACKGROUND: Endovascular treatment has been recognized as the first line therapy for renal artery aneurysm (RAA). However, RAA related with malignancies had been sporadically reported in the literature. Stent insertion should be contraindicated for RAAs with malignant etiology, whereas surgery be optimal. CASE PRESENTATION: A 40-year-old female underwent covered stent insertion to exclude the left RAA for suspected Takayasu arteritis in a reginal hospital. Three months later the RAA recurred with sign of threatened rupture, and the patient was transferred for salvage embolization with coils and thrombin injection. However, 20 days after the embolization procedure, multiple painful subcutaneous nodules developed in her flanks. Undifferentiated sarcoma was revealed by the pathological biopsy of the nodules. The RAA in this case was most likely related with the malignancy. CONCLUSION: Malignancy was the most likely etiology behind recurrent aneurysm in this case. Definite diagnosis is mandatory for interventional radiologists before stent insertion for treatment of RAA.

Protective effect of RNA interference targeting Ikkß on hepatic fibrosis in rats.

Nuclear factor-κB (NF-κB) is an important regulatory factor in cells. NF-κB has a wide range of biological activities. After activation, it participates in the transcription and regulation of many genes and plays a role in infection, inflammatory response, oxidative stress, cell multiplication, and apoptosis. The activation of the NF-κB signal pathway is dependent on the degradation of the IκB kinase ß (IKKß) complex. IKK ß is the key kinase in the NF-κB activation pathway. After inhibition, it can block the activation of NF-κB. IKKß is a key regulator of NF-κB activation, also an early regulator of inflammation in all stages of the immune response. This study aimed to investigate the effect of IKKß-siRNA lentivirus vector treatment for hepatic fibrosis of rats. An IKKß-siRNA expression plasmid was constructed and injected in the tail vein of rats. Then, IKKß-siRNA distribution in the liver was observed by immunofluorescence, and the quantitative polymerase chain reaction was used to detect inflammation-related and fibrosis-related factors. IKKß-siRNA lentiviruses could be delivered to the liver and significantly decrease carbon tetrachloride-induced hepatic fibrosis. Furthermore, serum transaminase levels significantly decreased, and inflammation-related and fibrosis-related factors decreased. IKKß-siRNA can be an effective method of anti-fibrosis gene therapy for liver fibrosis.

Application of Internet+-based Tsinghua PINS Remote Tech to improve sacral neuromodulation programming procedure.

OBJECTIVE: To explore the feasibility and safety of the Tsinghua PINS Remote Tech to facilitate sacral neuromodulation programming procedure. METHOD: For 22 patients who had previously participated in the phase III clinical trial for treating overactive bladder with the Tsinghua PINS sacral neuromodulation system during several Hospital, PINS Remote Tech was applied to perform postoperative parameter adjustment in order to evaluate the safety and reliability of this new technique. Telephone surveys on Remote Tech-related questionnaires were also conducted. RESULTS: 17/22 patients underwent 26 parameter adjustments, average adjustment frequency was 1.53 times per person; the average adjustment time was 23.4 ± 5.1 min (15-32 min). The total effective rate of the Remote control was 14/17 (82.3%). 7/17 (41.1%) patients' symptoms recurrence due to not knowing how to handle patient controller, these patients were instructed on how to use it correctly through Remote Tech even without reprogramming it. Other 10 patients received reprogramming. There was no discomfort during and after parameter adjustment. The questionnaire survey showed that the remote technology saved patients' time and lowered financial costs, significantly improved patient satisfaction. All patients expressed their willingness to recommend it to other patients. CONCLUSION: The PINS Remote Tech can significantly reduce the financial cost and provide a remote reprogram control service that is as safe and reliable as outpatient program control.

Room-temperature borylation and one-pot two-step borylation/Suzuki-Miyaura cross-coupling reaction of aryl chlorides.

A highly efficient room-temperature borylation strategy of aryl chlorides is described. Utilizing Buchwald's second-generation preformed catalyst, boronate esters were obtained for a wide range of substrates in high yield. The method was also applied to Suzuki-Miyaura cross-coupling reaction in a one-pot two-step sequential manner, providing a facile and convenient access to the direct synthesis of biaryl compounds from aryl chlorides.

Effects of Appropriate Prolonged Sacral Neuromodulation Testing in Improving Implantation Rate of a Permanent Implantable Pulse Generator in Patients with Refractory Lower Urinary Tract Dysfunctions in Mainland China.

BACKGROUND: Sacral neuromodulation (SNM) has become an effective method for treating lower urinary tract voiding dysfunction during the past 20 years. Because of the expensive cost, the number of implantable pulse generator (IPG) implantations per year in China is far lower than that in Western developed countries since 2012. This study was to summarize the effects of the appropriate prolonged SNM testing time in improving the implantation rate of a permanent IPG in patients with refractory lower urinary tract symptoms (LUTS) in mainland China. METHODS: From January 2013 to June 2016, 51 patients with refractory LUTS received SNM therapy. In this study, we compared the conversion rate 2 weeks after the Stage I test and final actual conversion rate. We also observed the complications (such as pain, infection, and electrode displacement) and effectiveness. We tried to improve an appropriate prolonged test time which was favorable for improving the SNM conversion rate while ensuring safety and effectiveness. RESULTS: Among 51 patients receiving SNM therapy, 19 patients (mean age 45.0 ± 16.9 years) had poor Stage I test results, and on an average, the electrode was removed 27.4 ± 9.6 days after the surgery. In one patient, the electrode was removed within 2 weeks; when the remaining 18 patients were questioned 2 weeks after testing, none of the patients wanted to terminate the test, and all the 18 patients desired to prolong the testing time to further observe the treatment effect. The remaining 32 patients (mean age 46.7 ± 15.3 years) received Stage II permanent implantation at 19.6 ± 10.4 days after the surgery. The overall Stage I-II conversion was 62.7% (32/51) in this study. Within 2 weeks after the surgery, only eight patients received Stage II permanent implantation, and the conversion rate was only 15.7% (8/51), which was much lower than the overall conversion rate of 62.7%. Nearly 84.4% (27/32) of the patients received Stage II implantation within 4 weeks. None of the patients had incision infections. In one patient, the entire system was removed 1 month after Stage II implantation due to pain in the implantation site. CONCLUSIONS: Appropriate extension of the Stage I testing time of an SNM-barbed electrode could significantly improve the Stage II permanent implantation rate in Chinese refractory LUTS patients; there were no wound infections, and the postoperative complication rate was low. This study recommended that Stage I period of SNM therapy should be 4 weeks according to safety and successful conversion rate.

Kidney transplantation after liver transplantation.

Kidney transplantation after liver transplantation (KALT) offers longer survival and a better quality of life to liver transplantation recipients who develop chronic renal failure. This article aimed to discuss the efficacy and safety of KALT compared with other treatments. The medical records of 5 patients who had undergone KALT were retrospectively studied, together with a literature review of studies. Three of them developed chronic renal failure after liver transplantation because of calcineurin inhibitor (CNI)-induced nephrotoxicity, while the others had lupus nephritis or non-CNI drug-induced nephrotoxicity. No mortality was observed in the 5 patients. Three KALT cases showed good prognoses, maintaining a normal serum creatinine level during entire follow-up period. Chronic rejection occurred in the other two patients, and a kidney graft was removed from one of them. Our data suggested that KALT is a good alternative to dialysis for liver transplantation recipients. The cases also indicate that KALT can be performed with good long-term survival.

[Expression and role of vascular endothelial growth inhibitor in sporadic clear cell renal cell carcinoma].

OBJECTIVE: To evaluate the expression of vascular endothelial growth inhibitor (VEGI) in sporadic clear cell renal cell carcinoma (CCRCC) and explore its relationships between VEGI expression, pathologic grade and tumor staging. METHODS: Western blot and immunohistochemical staining were used to detect the expression of VEGI in CCRCC cell line (786-O cells), CCRCC and paired normal kidney tissues. A total of 50 CCRCC cases were recruited. There were 37 males and 13 females with an average age of 53 ± 12 years. The tumor sizes were < 7 cm (n = 33) and ≥ 7 cm (n = 17). Their pathologic grades were G1 (n = 14), G2 (n = 22) and G3 (n = 14) and pathologic stages pT1 (n = 32), 10 pT2 (n = 10) and pT3 (n = 8). RESULTS: VEGI protein was predominantly located in cytoplasm. Compared with normal kidney tissues(mean optic density (MOD) of VEGI staining: 0.40 ± 0.16), it was lower in CCRCC tissues (MOD: 0.11 ± 0.06, P < 0.01). In addition, the positive rate of VEGI expression, the expression intensity and the MOD of VEGI protein were negatively correlated with the pathologic grade of CCRCC (r = -0.640, P < 0.01; r = -0.831, P < 0.01; r = -0.781, P < 0.01 respectively). The MOD of VEGI expression in ≥ 7 cm tumors (MOD, 0.08 ± 0.04) was significantly lower than that in < 7 cm tumors (MOD: 0.12 ± 0.06, P < 0.05). However, there was no correlations between the VEGI protein level and age, gender and pathologic stage of patients (P > 0.05). CONCLUSION: VEGI protein is predominantly located in cytoplasm. Compared with CCRCC tissues, VEGI protein level is higher in normal ones. In consideration of negative correlations between VEGI expression, pathologic grade and tumor size, it is implied that VEGI may play a negative regulatory role in the occurrence and development of CCRCC.