Rapid production of COVID-19 subunit vaccine candidates and their immunogenicity evaluation in pigs.

The emergence of various variants of concern (VOCs) necessitates the development of more efficient vaccines for COVID-19. In this study, we established a rapid and robust production platform for a novel subunit vaccine candidate based on eukaryotic HEK-293 T cells. The immunogenicity of the vaccine candidate was evaluated in pigs. The results demonstrated that the pseudovirus neutralizing antibody (pNAb) titers reached 7751 and 306 for the SARS-CoV-2 Delta and Omicron variants, respectively, after the first boost. Subsequently, pNAb titers further increased to 10,201 and 1350, respectively, after the second boost. Additionally, ELISPOT analysis revealed a robust T-cell response characterized by IFN-γ (171 SFCs/106 cells) and IL-2 (101 SFCs/106 cells) production. Our study demonstrates that a vaccine candidate based on the Delta variant spike protein may provide strong and broad protection against the prototype SARS-CoV-2 and VOCs. Moreover, the strategy for the efficient and stable expression of recombinant proteins utilizing HEK-293 T cells can be employed as a universal platform for future vaccine development.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

Nonsmoking and Nondrinking Oral Squamous Cell Carcinoma Patients: A Different Entity.

OBJECTIVE: Our goal was to analyze the demographic and pathologic characteristics as well as prognosis in nonsmoking and nondrinking (NSND) oral squamous cell carcinoma (SCC) patients compared with typical oral SCC patients. PATIENTS AND METHODS: A total of 353 patients were retrospectively enrolled and divided into two groups: the NSND group and the current smoking/current drinking (CSCD) group. Demographic, pathologic, and molecular data were compared between the two groups. The main research endpoints were locoregional control (LRC) and disease-specific survival (DSS). RESULTS: In the NSND group, 16.3%, 41.9%, and 53.5% of patients were aged no more than 40 years, were female, and had an educational background of high school or above compared to 3.7%, 6.0%, and 38.2% of patients in the CSCD group, respectively. A total of 15.1% of the NSND patients had SCC of the lower gingiva and floor of the mouth, which was lower than the 35.6% of patients in the CSCD group. CSCD patients were likely to have an advanced disease stage (48.7% vs 32.5%, p=0.042) and poorly differentiated cancer (26.6% vs 16.3%, p=0.042). The NSND patients had a mean Ki-67 index of 24.5%, which was lower than the mean of 35.7% in the CSCD patients. The two groups had no HPV infection and similar p16 expression (4.7% vs 10.1%, p=0.132), but there was higher expression of p53 (38.6% vs 17.4%, p<0.001) and p63 (59.9% vs 29.1%, p<0.001) in the CSCD group. The 5-year LRC rates for NSND patients and CSCD patients were 48% and 38%, respectively, and the difference was significant (p=0.048). The 5-year DSS rates for NSND patients and CSCD patients were 56% and 39%, respectively, and the difference was significant (p=0.047). Further, a Cox model confirmed the independence of smoking and drinking status for affecting LRC and DSS. CONCLUSION: NSND oral SCC patients are a different entity. HPV infection has a limited role in carcinogenesis in NSND patients, and p16 expression is associated with worse locoregional control.

[Paraquat poisoning mechanism and its clinical treatment progress].

Paraquat (PQ) as a kind of sterile and herbicides, has effects of contact-kill and systemic action, which can be absorbed quickly by green plants and make them wither and die. Therefore, it is widely used in the agricultural production and occupies a large part in our country's pesticide market. PQ poisoning has become one of the most common pesticide poisoning in our country. PQ can be passivated when combining with soil, but it has great toxicity for human. There are still no specific antidotes for PQ poisoning at home and abroad, and the death rate of PQ oral poisoning is up to 95%. Clinically comprehensive treatment is adopted, including gastric lavage, intentional diarrhea, diuresis and blood perfusion. However, the therapeutic effect is not good and the case fatality rate keeps high. It has become one of the hot issues for emergency medicine study to search PQ's special efficiency measures. This paper briefly reviews PQ's poisoning mechanism and its clinical treatment progress to provide new exploration direction and treatment ideas for basic research and clinical treatment of PQ.

[Evaluation of adsorption effect of activated charcoal on oral paraquat poisoning: an experimental study on large animal].

OBJECTIVE: To study the adsorption effect of activated charcoal suspension on paraquat (PQ) in gastrointestinal tract of beagles exposed to PQ. METHODS: Twenty healthy male beagles were randomly divided into experimental group and control group, with 6 beagles in each group. 20% PQ solution (a dose of 30 mg/kg) was prescribed through stomach for beagles in both groups. After exposure to PQ for 30 minutes, the beagles in experimental group were given activated charcoal suspension (1.0 g/kg of type I activated charcoal powder mixed with 100 mL of normal saline) by gavage, while the control group was only given equal volume of normal saline. After exposure to PQ for 10 minutes, 30 minutes, and 1, 2, 4, 8, 12, 24, and 48 hours, blood was collected from hepatic portal veins and peripheral veins to detect the PQ concentration change in the plasma. The toxicokinetics software DAS 2.1.1 was applied to analyze PQ concentration and compare the change in toxicokinetics parameters between the both groups. The change in vital signs including heart rate (HR), respiratory rate (RR) and pulse oxygen saturation (SpO2) was dynamically monitored 10 minutes before exposure, 4 hours and each day from the 1st to the 7th day after exposure. RESULTS: After exposure to PQ, the poison concentration in the plasma of hepatic portal veins and peripheral veins in the control group rose quickly and reached peak 4 hours later. It fell quickly at first, and fell slowly 8 hours later. But in the experimental group, the increase rate to the peak was significantly slow. Besides, PQ peak fell more obviously than that in the control group and it was about 50% of the control group (µg/L: 123.50±11.67 vs. 255.18±12.29 in blood from hepatic portal veins, 122.35±11.72 vs. 250.86±11.15 in blood from peripheral veins). After 8 hours it fell much more quickly than that of the control group. After exposure to PQ for 48 hours, PQ concentration in the plasma was still lower than that of the control group (µg/L: 0.53±0.18 vs. 15.98±5.58 in blood from hepatic portal veins, 0.31±0.01 vs. 15.03±4.82 in blood from peripheral veins, both P < 0.01). With the toxicokinetics analysis, compared with the control group, the maximum concentration (Cmax) and area under the curve (AUC) of PQ in the plasma of hepatic portal veins and peripheral veins in the experimental group were significantly decreased [Cmax (µg/L): 125.07±9.49 vs. 255.18±12.29 in blood from hepatic portal veins, 123.38±9.52 vs. 250.86±11.15 in blood from peripheral veins; AUC (mg×L-1×h-1): 1.6±0.2 vs. 3.3±0.4 in blood from hepatic portal veins, 1.5±0.2 vs. 3.2±0.3 in blood from peripheral veins], time to the peak (Tmax) of PQ was slowed (hours: 5.3±1.9 vs. 4.0±0.0 in blood from hepatic portal veins, 4.7±1.5 vs. 4.0±0.0 in blood from peripheral veins), and PQ plasma half-life (t1/2) and mean retention time (MRT) were significantly shortened [t1/2 (hours): 3.8±1.2 vs. 15.4±3.7 in blood from hepatic portal veins, 3.5±1.0 vs. 15.5±2.7 in blood from peripheral veins; MRT (hours): 8.0±1.5 vs. 13.4±1.2 in blood from hepatic portal veins, 7.6±1.3 vs. 13.3±1.2 in blood from peripheral veins; all P < 0.01]. After exposure to PQ, HR and RR in both the experimental group and the control group increased and reached to the peak about the 4th day and then the increase rate began to slow down gradually; SpO2 slowed down gradually and reached to the valley about the 4th day and then it began to recover, but the change range of vital signs in the experimental group was smaller than that of the control group, and the parameters were significantly better than those of control group [4-day HR (bpm): 134.50±3.04 vs. 142.00±6.43, 4-day RR (times/min): 31.00±0.58 vs. 34.33±0.94, 4-day SpO2: 0.900±0.006 vs. 0.873±0.005, all P < 0.05]. CONCLUSIONS: Activated charcoal administrated at 30 minutes after PQ poisoning can slow down the increase rate of PQ concentration in the plasma, decrease the peak concentration and has less influence on vital signs in beagles.

Energy metabolism regulated by HDAC inhibitor attenuates cardiac injury in hemorrhagic rat model.

A disturbance of energy metabolism reduces cardiac function in acute severe hemorrhagic patients. Alternatively, adequate energy supply reduces heart failure and increases survival. However, the approach to regulating energy metabolism conductive to vital organs is limited, and the underlying molecular mechanism remains unknown. This study assesses the ability of histone deacetylase inhibitors (HDACIs) to preserve cardiac energy metabolism during lethal hemorrhagic injury. In the lethally hemorrhagic rat and hypoxic myocardial cells, energy metabolism and heart function were well maintained following HDACI treatment, as evident by continuous ATP production with normal cardiac contraction. Valproic acid (VPA) regulated the energy metabolism of hemorrhagic heart by reducing lactate synthesis and protecting the mitochondrial ultrastructure and respiration, which were attributable to the inhibition of lactate dehydrogenase A activity and the increased myeloid cell leukemia-1 (mcl-1) gene expression, ultimately facilitating ATP production and consumption. MCL-1, the key target of VPA, mediated this cardioprotective effect under acute severe hemorrhage conditions. Our results suggest that HDACIs promote cardioprotection by improving energy metabolism during hemorrhagic injury and could therefore be an effective strategy to counteract this process in the clinical setting.

Valproic acid-mediated myocardial protection of acute hemorrhagic rat via the BCL-2 pathway.

BACKGROUND: Hemorrhage is a major cause of morbidity and mortality among trauma patients. The pathophysiologic changes following acute severe hemorrhage and tissue hypoxia lead to an imbalance of protein acetylation. Histone deacetylase inhibitors (HDACIs) were reported to restore the acetylation imbalance and serve as potential drugs for treating severe hemorrhage. However, the molecular mechanism of HDACI-mediated cytoprotection remains unclear. In this study, we examined the myocardial protective effects and respective mechanism of the HDACI valproic acid (VPA) administered during hemorrhagic and hypoxic stress in vivo and in vitro. METHODS: In vivo, the therapeutic effect of VPA was evaluated in acute severe hemorrhagic rats, and the expressions of BCL-2 signal pathway molecules were observed in rat heart tissues. To explore the molecular mechanism of VPA-mediated myocardial protection, a cobalt chloride (CoCl2)-induced hypoxia model of rat H9c2 cardiomyoblasts was applied to mimic hypoxic injury raised by acute hemorrhage. RESULTS: VPA administration significantly improved the 4-hour survival rate of hemorrhagic animals from 55% to 100% and protected H9c2 cells against CoCl2-induced hypoxic injury at a dose of between 12.5 µM and 100 µM. Increased expression of BCL-2 messenger RNA was observed following VPA treatment in the heart tissues of hemorrhagic rats (approximately 4.9-fold) and in H9c2 cells that survived CoCl2-induced hypoxia (approximately 4.9-fold). Western blot analysis showed a concomitant increase in BCL-2 protein expression and Akt phosphorylation following VPA treatment. The cytoprotective activity of VPA was diminished by triciribine-mediated inhibition of Akt activation and by silencing of BCL-2 gene expression. CONCLUSION: These findings suggest that VPA protects myocardial cells from hemorrhagic and hypoxic stress through the Akt/BCL-2 survival pathway, indicating a potential use of HDACIs for acute severe hemorrhage treatment.

Hypertonic Saline Dextran Ameliorates Organ Damage in Beagle Hemorrhagic Shock.

OBJECTIVE: The goal of this study was to investigate the effect of hypertonic saline with 6% Dextran-70 (HSD) resuscitation on organ damage and the resuscitation efficiency of the combination of HSD and lactated ringers (LR) in a model of hemorrhage shock in dogs. METHODS: Beagles were bled to hold their mean arterial pressure (MAP) at 50 ± 5 mmHg for 1 h. After hemorrhage, beagles were divided into three groups (n = 7) to receive pre-hospital resuscitation for 1 h (R1): HSD (4 ml/kg), LR (40 ml/kg), and HSD+LR (a combination of 4 ml/kg HSD and 40 ml/kg LR). Next, LR was transfused into all groups as in-hospital resuscitation (R2). After two hours of observation (R3), autologous blood was transfused. Hemodynamic responses and systemic oxygenation were measured at predetermined phases. Three days after resuscitation, the animals were sacrificed and tissues including kidney, lung, liver and intestinal were obtained for pathological analysis. RESULTS: Although the initial resuscitation with HSD was shown to be faster than LR with regard to an ascending MAP, the HSD group showed a similar hemodynamic performance compared to the LR group throughout the experiment. Compared with the LR group, the systemic oxygenation performance in the HSD group was similar but showed a lower venous-to-arterial CO2 gradient (Pv-aCO2) at R3 (p < 0.05). Additionally, the histology score of the kidneys, lungs and liver were significantly lower in the HSD group than in the LR group (p < 0.05). The HSD+LR group showed a superior hemodynamic response but higher extravascular lung water (EVLW) and lower arterial oxygen tension (PaO2) than the other groups (p < 0.05). The HSD+LR group showed a marginally improved systemic oxygenation performance and lower histology score than other groups. CONCLUSIONS: Resuscitation after hemorrhagic shock with a bolus of HSD showed a similar hemodynamic response compared with LR at ten times the volume of HSD, but HSD showed superior efficacy in organ protection. Our findings suggest that resuscitation with the combination of HSD and LR in the pre-hospital setting is an effective treatment.

Chidamide, a novel histone deacetylase inhibitor, synergistically enhances gemcitabine cytotoxicity in pancreatic cancer cells.

Pancreatic cancer is a lethal human malignancy with an extremely poor prognosis and urgently requires new therapies. Histone deacetylase inhibitors (HDACIs) represent a new class of anticancer agents and have shown promising antitumor activities in preclinical models of pancreatic cancer. In this study, we sought to determine the antitumor effects of a novel HDACI, chidamide (CS055), in pancreatic cancer cells alone or in combination with gemcitabine. Treatments of BxPC-3 or PANC-1 pancreatic cancer cell lines with chidamide resulted in dose- and time-dependent growth arrest, accompanied by induction of p21 expression. When combined in a sequential schedule, chidamide synergistically enhanced gemcitabine-induced cell growth arrest and apoptosis, accompanied by cooperative downregulation of Mcl-1 and loss of mitochondrial membrane potential (ΔΨm). Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression. Our results suggest that chidamide has a therapeutic potential for treating pancreatic cancer, especially in combination with gemcitabine.

[Detoxification effects of two drugs in thallium -poisoned mice].

OBJECTIVE: To observe the thallium eliminating effect of prussian blue, pentetate zinc trisodium (Zn-DTPA), and their combined use in the treatment of acute thallium poisoning in mice. METHODS: Thallium poisoned mice were reproduced by oral administration of 0.2 ml thallous nitrate (3 mg/ml). They were assigned randomly to four groups according to the random number table method, namely, model group, prussian blue group, Zn-DTPA group and the combination therapy group, with 10 mice in each group. Prussian blue was administered orally [4.52 g×kg(-1)×d(-1), total four times], and Zn-DTPA was injected intraperitoneally [500 mg×kg(-1)×d(-1), one time]4 hours after giving thallium. The dosage of both drugs in combination treatment was as the same as described above. After treatment for 5 days, all the animals were sacrificed. Brain, intestine, kidney and liver of 1 mouse from each group were collected for pathological examination to observe the necrosis. Thallium contents of blood, brain, urine and feces from the other mice were determined. RESULTS: Pathological examination showed that the damage to intestine, kidney and liver was less obvious in treatment group compared with those of the model group. The effect was most obvious in the combination treatment group. However, brain damage was slightly improved. Thallium content in blood (mg/ml) of prussian blue group and the combination treatment group decreased obviously compared with the model group, and the decrease was more obvious in the combination treatment group (0.05 ± 0.01 vs. 0.18 ± 0.02). Thallium content in urine (mg/ml) and feces (mg/kg) was significantly increased after treatment, and the thallium elimination was most significant in the combined treatment group (urine: 11.34 ± 0.81 vs. 0.02 ± 0.01, feces: 13.11 ± 1.84 vs. 0.21 ± 0.07, both P < 0.01). Thallium content in brain was similar among all the groups. CONCLUSIONS: The single and combined use of prussian blue and Zn-DTPA could reduce the damage in intestine, kidney and liver. Combined use of prussian blue and Zn-DTPA for acute thallium poisoning mice is more efficacious than single use of either drug.

[Studying the therapeutic effects of hemoperfusion with continuous venovenous hemofiltration on the patients with acute paraquat poisoning].

OBJECTIVE: To explore the therapeutic effects of hemoperfusion (HP) with continuous venovenous hemofiltration (CVVH) on the patients with acute paraquat poisoning. METHODS: Ninety-one patients with acute paraquat poisoning were randomly divided into HP group (49 cases) and HP-CVVH group (42 cases). The mortality, survival duration and the death causes between the two groups were compared and analyzed. RESULTS: There were no significant differences in mortality (59.2% versus 61.9%) between the two groups. The mean time between poisoning and death in HP-CVVH group was (4.9 +/- 3.1) days, which was significantly longer than that (3.5 +/- 2.0) days in HP group (P < 0.05). The death proportion on 4th day after poisoning in HP group was 62.1% (18/29), which was significantly higher than that (30.8%, 8/26) in HP-CVVH group (P < 0.05). The hypoxia appeared in 4.3 +/- 2.5 days after poisoning in HP-CVVH group, which was significantly longer than that (3.2 +/- 1.9) days in HP group (P < 0.05). The mortality due to respiratory failure in HP group was 20.4% (10/49), which was significantly lower than that (40.5%, 17/42) in HP-CVVH group (P < 0.05). The incidence of acute renal failure in HP group was 63.3% (31/49), which was significantly higher than that (40.5%, 17/42) in HP-CVVH group (P < 0.05). CONCLUSION: The combined therapy of HP and CVVH can prevent the patients with acute paraquat poisoning from early death and prolong the survival duration, but can not reduce mortality for the patients with acute paraquat poisoning.

[Study on the prognosis of patients with acute paraquat intoxication].

OBJECTIVE: To study the correlation factors of acute paraquat intoxication prognosis. METHODS: The early paraquat concentration in plasma and urine, leukocyte count, hepatic and renal function, amylase, electrolyte and the parameters of arterial blood gas were analyzed retrospectively in 111 patients with acute paraquat intoxication. RESULTS: 43 cases (38.7%) of all the 111 patients survived and the other 68 cases (61.3%) died. The patient, whose paraquat concentration was not more than 8.0 µg/ml in plasma and 276.0 µg/ml in urine, could survive. But some patients could die, only if there was no paraquat found in plasma. The paraquat levels in plasma and urine were significantly lower in survivors [(0.82 ± 1.70), (28.12 ± 51.17) µg/ml] than in nonsurvivors [(9.32 ± 12.04), (384.53 ± 597.93) µg/ml, respectively] (P < 0.01). The levels of leukocyte count, serum creatinine, aspartate aminotransferase (AST), and amylase were significantly higher in nonsurvivors than in survivors (P < 0.05, P < 0.01). In addition, metabolic acidosis was easier to appear in nonsurvivors. The multiple logistic regression analysis indicated that the paraquat concentration in plasma and urine, leukocyte count, creatinine and base excess were all related to survival. CONCLUSION: The higher paraquat concentration in plasma and urine, leucocytosis, renal dysfunction and metabolic acidosis are all important factors for the prognosis of paraquat intoxication.

[The diagnostic value of butyrylcholinesterase in acute organophosphorus pesticide poisoning].

OBJECTIVE: To assess the value of butyrylcholinesterase (BuChE) in the diagnosis of acute organophosphorus pesticide poisoning (AOPP), and to investigate the relationship between the activity of whole blood acetylcholinesterase (AChE) and plasma BuChE in patients of AOPP in order to re-evaluate the diagnostic value of BuChE. METHODS: An improved Ellman method was employed to determine the activity of AChE and BuChE in 21 AOPP inpatients due to different kinds of pesticides (from April to September in 2009) during the course of the illness, with 26 healthy volunteers as normal control. The relationship between BuChE activity and other clinical indicators of 113 inpatients (from January 2008 to April 2009) was also retrospectively analyzed. RESULTS: The normal value of AChE and BuChE were (105 + or - 33) U/Hb and (15 807 + or - 3 495) U/L . The inhibition levels of these two enzymes were different. When the AChE activity was lower than 50%, 30% or 20%, the activity of BuChE was lower than 20%, 10% or 5% correspondingly. The tendency of changes in the two enzymes was similar and coincided well with the clinical symptoms after poisoning. The results of sequential detection showed that a significant decrease or persistent inhibition of BuChE activity by less than 5% indicated a high level of organophosphorus pesticide in the body. However, an elevation of BuChE indicated a favorable outcome. CONCLUSION: BuChE is one of the ideal diagnostic and classification criteria for AOPP. When the inhibition level of BuChE reaches 20%, AOPP is of moderate degree, when it reaches 10%, severe AOPP can be diagnosed, with different kinds of organophosphorus pesticides taken into consideration.