Objective To rescue a recombinant oncolytic influenza virus chimeric with IL-21 and evaluate its inhibitory effects and safety against hepatocell ular carcinoma(HCC)both in vitro and in vivo,and to explore the mechanism by which this virus enhances antitumor effects when combined with anti-programmed cell death protein 1(anti-PD-1)antibody.Methods The IL-21 gene fragment was inserted into the nonstructural protein(NS)sequence of the influenza virus PR8 using reverse genetics(RG)technology to rescue the recombinant oncolytic influenza virus rOV-IL-21-NS.The virus titer and virulence were determined using the 50％tissue culture infectious dose(TCID50)and hemagglutination assays.The successful insertion of the exogenous gene into the NS sequence was verified using RT-qPCR,gel electrophoresis,and sequencing analysis.Viral morphology and size were observed with transmission electron microscopy.The impact of the virus on the viability of hepatocellular carcinoma cells was assessed with CCK-8 assay.A subcutaneous tumor model of HCC was established in 45 female C57BL/6 mice(8 weeks old,weighing 16～20 g),and then the mice were randomly assigned into PBS,PR8,anti-PD-1,rOV-IL-21-NS,and the rOV-IL-21-NS+anti-PD-1 treatment groups,with 9 mice in each group,to evaluate the anti-tumor effects of monotherapy versus combination therapy.Flow cytometry was conducted to assess the regulatory effects of monotherapy and combination therapy on the tumor immune microenvironment.Results RG technology successfully rescued the recombinant oncolytic influenza virus rOV-IL-21-NS.Sequencing confirmed the successful insertion of IL-21 into the target sequence,and the obtained virus could be stably propagated,with its sixth passage reaching a hemagglutination titer of 211,and a viral titer of 6 Log10(TCID50/mL).Oncolytic virus rOV-IL-21-NS,at a multiplicity of infection(MOI)of 3,selectively reduced the viability of HCC cells without significantly affecting normal liver cells.Compared to the control group,the combination of rOV-IL-21-NS and anti-PD-1 antibodies significantly inhibited tumor growth(P＜0.001)and increased the proportions of CD4+T and CD8+T cells in the spleen tissue of the mouse model of subcutaneous HCC tumor(P＜0.001).Conclusion The recombinant oncolytic influenza virus rOV-IL-21-NS chimeric with IL-21 can effectively and safely exert targeted killing to HCC cells,enhance T cell activation by synergizing with anti-PD-1 antibodies,and improve the immune microenvironment.

Objective To explore the influence of negative lymph node count (NLNC) on the prognosis of patients with gastric signet ring cell carcinoma (GSRC) and develop a prognostic nomogram based on NLNC. Methods On the basis of the SEER database, 2 101 patients diagnosed with GSRC were collected and randomly divided into the modeling group and validation group to test the relationship between clinicopathological characteristics and the prognosis of GSRC. The multivariate Cox proportional hazard regression model was used to analyze the independent risk factors affecting overall survival and establish a prognostic prediction model. The consistency index (C-index), calibration curve, net reclassification index (NRI), integrated discrimination improvement (IDI), and decision curve analysis (DCA) were used to evaluate the accuracy and clinical applicability of the nomogram. Results All patients were divided according to the ratio of 7:3, with 1 473 in the modeling group and 628 in the validation group. NLNC > 10 (HR=0.578, 95%CI: 0.504-0.662, P < 0.001) was a protective factor for the prognosis of patients with GSRC, and the nomogram model was established based on multivariate Cox proportional hazards model. The C-index values of the nomogram were 0.737 (95%CI: 0.720-0.753) and 0.724 (95%CI: 0.699-0.749) in the modeling and validation groups, respectively, showing good discrimination. The calibration curves showed high consistency of the model. NRI=17.77%, continuous NRI=36.34%, and IDI=4.2% indicated that the model had positive returns compared with the traditional model. The DCA was far from the baseline, indicating that the model had good clinical applicability. Conclusion The increase in NLNC is a favorable factor for the prognosis of patients with GSRC, and a relatively accurate nomogram was established to predict the prognosis of patients with GSRC and help clinicians conduct individualized prognostic evaluations.

Objective:To summarize and analyze the clinical data of Chinese patients with colony-stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy, and clarify the phenotypic and genetic characteristics of Chinese patients.Methods:Medical history of patients with CSF1R-related leukoencephalopathy diagnosed from April 1, 2018 to January 31, 2021 in the department of neurology of 22 hospitals in China was collected, and scores of Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment Scale (MoCA), magnetic resonance severity scale were evaluated. Group comparison was performed between male and female patients.Results:A total of 62 patients were included, and the male-female ratio was 1∶1.95. The age of onset was (40.35±8.42) years. Cognitive impairment (82.3%, 51/62) and motor symptoms (77.4%,48/62) were the most common symptoms. The MMSE and MoCA scores were 18.79±7.16 and 13.96±7.23, respectively, and the scores of two scales in male patients (22.06±5.31 and 18.08±5.60) were significantly higher than those in females (15.53±7.41 , t=2.954, P=0.006; 10.15±6.26, t=3.328 , P=0.003). The most common radiographic feature was bilateral asymmetric white matter changes (100.0%), and the magnetic resonance imaging severity scale score was 27.42±11.40, while the white matter lesion score of females (22.94±8.39) was significantly higher than that of males (17.62±8.74 , t=-2.221, P<0.05). A total of 36 CSF1R gene mutations were found in this study, among which c.2381T>C/p.I794T was the hotspot mutation that carried by 17.9% (10/56) of the probands. Conclusions:The core phenotypic characteristics of CSF1R-related leukoencephalopathy in China are progressive motor and cognitive impairment, with bilateral asymmetrical white matter changes. In addition, there exist gender differences clinically, with severer cognitive impairment and imaging changes in female patients. Thirty-six CSF1R gene mutations were found in this study, and c.2381T>C/p. I794T was the hotspot mutation.

Objective:To investigate the effect of edaravone, a free radical scavenger, on the regulation of retinal autophagy and the protection of photoreceptor cells at the early stage of experimental retinal detachment (RD) in rats.Methods:Fifty-one adult male Sprague-Dawley rats were used for RD model establishment, and another 24 rats were served as PBS injection group.The RD model was established via subretinal injection of 0.5% sodium hyaluronate into the right eye of the rats and the rats with successful modeling were randomly divided into RD model group and edaravone treatment group.The rats in the edaravone treatment group were given edaravone of 3 mg/kg intraperitoneally, twice a day after modeling, and the rats in the PBS injection group and RD model group were given equal volume of normal saline.Rats were sacrificed on the 1st day, 3rd day and 7th day following modeling.The T-superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) content in the intraocular fluid was detected.The expression levels of superoxide dismutase 2 (SOD2), nuclear factor E2-related factor 2 (Nrf2), autophagy related gene 4 (Atg4), microtubule-associated protein 1 light chain 3B (LC3B) and other proteins in retinal tissue were identified by Western blot analysis.TUNEL staining was performed on paraffin sections of the whole eyeball to analyze the apoptosis of photoreceptor cells.The study protocol was approved by an Ethics Committee of Xi'an Fourth Hospital (No. 2016016). The use and care of animals complied with the Regulations on the Administration of Experimental Animals.Results:The RD area was more than 60% in rat eyes of RD model.There were significant differences in MDA content and T-SOD activity among different groups at various time points (MDA: Fgroup=385.513, P<0.01; Ftime=13.021, P<0.01.T-SOD: Fgroup=48.865, P<0.01; Ftime=7.700, P=0.003). Compared with the PBS injection group, the MDA concentration was significantly increased and the T-SOD activity was significantly decreased in the RD group and edaravone treatment group on the 1st, 3rd and 7th day after modeling (all at P<0.05). The MDA concentration was significantly reduced and the T-SOD activity was significantly elevated in the edaravone treatment group on the 1st, 3rd and 7th day after modeling in comparison with those of the RD group (all at P<0.05). Compared with the PBS injection group, the relative expression levels of SOD2 and Nrf2 proteins were significantly increased in the RD group and edaravone treatment group on the 1st, 3rd and 7th day after modeling (all at P<0.05), and Atg4 and LC3B-Ⅱ/LC3B-Ⅰ were significantly increased on the 1st, 3rd and 7th day after modeling (all at P<0.05). The expression level of SOD2 in the edaravone treatment group was significantly higher than that in the RD group on the 1st, 3rd and 7th day after modeling (all at P<0.05), and the expression level of Nrf2 was significantly increased in the edaravone treatment group on the 1st and 3rd day after modeling compared with that of the RD group (both at P<0.05), and the expression levels of Atg4 and LC3B-Ⅱ/LC3B-Ⅰ were significantly increased in the edaravone treatment group on the 3rd day after modeling in comparison with those of the RD group (both at P<0.05). No significant TUNEL positive cells were observed in PBS injection group at all time points, and TUNEL positive cells were observed on the 1st, 3rd and 7th day after modeling in the RD group, and the expression level of caspase-3 in the RD group was significantly increased in comparison with that of the PBS injection group ( P<0.05). The apoptosis of photoreceptor cells and the expression level of caspase-3 in edaravone treatment group were significantly decreased in comparison with those of the RD group on the 1st, 3rd and 7th day after modeling (all at P<0.05). Conclusions:The intraperitoneal injection of edaravone, twice a day, can significantly improve the antioxidant capacity of the retina after experimental RD in rats, regulate retinal autophagy and reduce the apoptosis of photoreceptor cells in early-stage RD.

Objective To explore the effect and mechanism of local administration of thymosin β4 (Tβ4) on bone regeneration in a distraction osteogenesis model of rat fenur.Methods Sixty Sprague-Dawley rats were randomly divided into groups A,B and C in this study.A distraction osteogenesis model was established in the left femur after osteotomy.At the end of distraction period,the bone regeneration area in group A was subjected to no treatment,that in group B to injection of phosphate buffer saline (PBS),and that in group C to injection of Tβ4.On days 22,29 and 43 postoperatively,the rats from each group were randomly sacrificed and processed for observation of bone regeneration in the distraction osteogenesis area using radiography,Micro-CT,histology and immunohistochemical staining.RT-PCR was used to detect the expression of related genes as well.Results Radiography revealed that the bone regeneration in group C was superior to that in groups A and B on days 22,29 and 43 postoperatively.Micro-CT examination showed significantly increased bone volume (BV),bone mineral density (BMD) and ratio of bone volume to tissue volume (BV/TV) in group C on days 22,29 and 43 postoperatively,and significantly decreased ratio of bone surface area to bone volume on postoperative day 43 in comparison with groups A and B(P ＜ 0.05).HE staining indicated that local capillary density was significantly higher in group C than in groups A and B after local administration of Tβ4 in the distraction osteogenesis.Immunohistochemical staining showed that the capillary density and osteoblasts in group C were higher than in groups A and B.RT-PCR results revealed significantly higher expression of eNOS and Osterix mRNA in the local callus in group C on postoperative day 22 than in groups A and group B (P ＜ 0.05).Conclusion Local administration of thymosin β4 may promote bone formation,which is probably related to the increased expression of eNOS and Osterix.

Objective To investigate the nutritional management level of Department of Clinical Nutrition in elderly inpatients through analyzing the actual hospital dietary intakes in elderly inpatients in Peking Union Medical College Hospital from 2013-2015.Methods Using continuous sampling, the nutrients contents and the eating rates of hospital meals ordered by the elderly inpatients on the 1st, 15th, 29th of every month from May 2013 to October 2015 were selected.The intake levels of energy, protein, calcium, vitamin A, vitamin B1 , and vitamin C were calculated and compared with the Chinese dietary reference intakes (DRIs).The nutritional in-take differences between diabetes diet and the basic hospital diet were also compared.Results A total of 90 days of hospital dietary data from 8 402 elderly inpatients were included in the study.The average eating rate was (50.1 ±4.2)%, which was lower than the general eating rate of the hospital [ (59.0 ±4.0)%, P<0.001]. Only protein intake from hospital diet reached the requirement target [male (103.1 ±47.3)%, femal (98.3 ± 33.8)%].Individual compliance rates were 63.2%and 59.8%, respectively, while the other kinds of nutrients were lower.The compliance rates of energy [ (73.3 ±26.3)%vs.(62.1 ±38.2)%, P<0.001] and ma-jor nutrient intake [ protein: ( 119.1 ±41.2 )% vs.( 93.3 ±65.1 )%, P <0.001; calcium: ( 55.5 ± 26.7)% vs.(34.3 ±34.2)%, P <0.001; vitamin A: (75.2 ±48.3)% vs.(57.4 ±97.1)%, P<0.001;vitamin B1:(76.4 ±38.2)%vs.(52.1 ±46.6)%, P<0.001;vitamin C:(92.2 ±51.4)%vs. (49.3 ±55.0)%, P<0.001) in diabetes diet group were significantly higher than those in the basic hospi-tal diet group.Conclusions The nutritional intakes from hospital diets may not meet the nutritional require-ments based on DRIs in elderly inpatients.Medical diet designed by physicians and dietitians should be ex-panded to improve the nutritional management level for these patients.

Objective To determine the levels of blood pancreatic stone protein/ regenerating protein(PSP/reg)and to explore the value of PSP/ reg in assessing severity and predicting prognosis of sepsis with or without panc-reatic damage. Methods In this prospective study,the clinical data and blood samples to measure PSP/ reg levels in 1,3 and 7 days after admission in the Pediatric Intensive Care Unit,Hunan Children's Hospital,from the children with sepsis from September 2013 to September 2014 were collected. According to levels of pancreatic trypsin(amylase or li-pase),the children were divided into normal group,mildly elevated group(1 - 3 times),and severely elevated group (more than 3 times);based on the condition of sepsis,the children were divided into sepsis group and severe sepsis group. Chi - square test,nonparametric rank sum test,Spearman correlation and area under the receiver operating cha-racteristic curve(AUC),Kaplan - Meier survival curves were used for statistical analysis. Results (1)A total of 226 children with sepsis were included in this study,among them pancreatic trypsin was normal in 159 cases(70. 4% ), pancreatic trypsin mildly elevated in 44 cases( 19. 4% ),and pancreatic trypsin severely elevated in 23 cases (10. 2% );125 cases(55. 3% )were sepsis,and 101 cases(44. 7% ,of which 29 cases were sepsis shock)were severe sepsis.(2)The correlation between PSP/ reg and serum amylase was maximum,while PSP/ reg had some correlation with serum lipase,but there was no correlation with C - peptide,insulin,glucose.(3)When pancreatic trypsin was normal or mildly elevated,severe sepsis group had higher levels of PSP/ reg than that of sepsis group(Z = - 7. 627,- 2. 965,P =0. 000,0. 003);while pancreatic trypsin was severe elevated,there was no significant difference in PSP/ reg levels be-tween sepsis group and severe sepsis group(Z = - 0. 134,P = 0. 894).(4)The AUC of PSP/ reg and procalcitonin to assess severity of sepsis were greater than 0. 7 in the children whose pancreatic trypsin was normal or mildly elevated, and there was no significant difference between them(P ﹥ 0. 05);while the AUC of C - reactive protein and WBC were less than 0. 7.(5)PSP/ reg levels gradually increased in non - survival children in 3 and 7 days,while survival children gradually declined. The AUC of PSP/ reg to predict prognosis was 0. 775,and 60 μg/ L was viewed as the critical point, sensitivity 89% ,specificity 67% . Children with PSP/ reg levels ≤60 μg/ L had higher rate of survival than children with ﹥ 60 μg/ L(χ2 = 36. 874,P = 0. 000). Conclusions PSP/ reg is closely associated with pancreatic exocrine func-tion,but PSP/ reg still can assess severity and predict prognosis in children with sepsis and pancreatic damage.

OBJECTIVETo study the value of Pentraxin 3 (PTX3) in diagnosing the severity and cardiovascular function of the critically ill children. Method A total of 178 patients who were older than 28 days, with acute infection of respiratory or neurological system, excluding chronic or special disease, and admitted to the pediatric intensive care unit (PICU) of Hunan Children's Hospital from October 1, 2013 to April 30, 2014 were enrolled, including 102 male cases and 76 female cases. The ages ranged from 1 month to 13 years and 1 month, 78 of them were less than 1 year old ; 58 cases were between 1 to 3 years old; 42 cases were above 3 years old; 101 cases were diagnosed as respiratory system diseases, 77 cases had nervous system diseases. PTX3 was detected with enzyme-linked immunosorbent assay (ELISA) within 1 d after enrollment, at 3 days and 7 days, meanwhile, troponin, myocardial enzyme, brain-type natriuretic peptide (BNP), C-reactive protein (CRP), plasma calcitonin (PCT) and WBC etc. Were measured. According to the plasma PTX3 value which were measured within 24 h after enrollment the patients were divided into three groups: mildly elevated group (< 44 µg/L) 41 cases; moderately elevated group (44 - < 132 µg/L) in 66 cases; severely elevated group 71 cases (132 µg/L or higher). Those 178 patients were divided into 3 groups according to the degree of infection: non-sepsis group (78 cases), sepsis group (70 cases), severe sepsis group (30 cases), and in each group, those with heart failure were respectively 19 cases, 28 cases, 17 cases. Analysis of the plasma PTX3 expression changes in different clinical manifestations, different condition, different degrees of organ damages and prognosis for the patient. The continuous variables were analyzed with t-test, F-test, H-test, the categorical variables were analyzed with Chi-square test, and the correlation analysis was performed to calculate Pearson coefficients.

RESULTThe PTX3 value measured within 24 h after enrollment increased with the degree of infection (50. 4(35. 2,70. 4) µg/L; 175. 8 (99. 6, 309. 9) µg/L;419. 9 (168. 3, 468. 6) µg/L; H = 88. 345, P = 0. 000). PTX3 level gradually declined, while in severe sepsis group decreased slowly (P <0. 05); the area under the ROC curve of Plasma PTX3 was larger than that of other inflammatory markers such as CRP and PCT, white blood cells and neutrophils in the diagnosis of sepsis; while the former three are PTX3, PCT and CRP (the sensitivity and specificity respectively were 0. 77, 0. 68; 0. 66, 0. 6; 0. 47, 0. 55); the PTX3 value of the severely elevated group was significantly higher than those of the mildly and moderately elevated groups (P <0. 05). The proportion of having 3 or more organs failure increased as the PTX3 rising among the groups of mildly elevated group, moderately elevated group and severely elevated group (1(2. 4%), 4(6. 1%), 14(19. 7%) χ2 =16. 16,P = 0. 000); and in each group, the proportion of having good and poor prognosis for these three groups were different (33 (80.5%) and 8 (19. 5%), 35 (53%) and 31 (47%), 28 (39.4%) and 43 (60.6%), χ = 17. 663, P = 0. 000). The K-M curve for these three groups had statistically significant difference (χ2 = 7. 086, P = 0. 029). Those with heart failure had higher PTX3 value than those in non-heart failure at the same degree of infection. PTX3 value increased with myocardial enzyme (troponin, creatine kinase isoenzyme, BNP) levels. In the diagnosis of heart failure, the area under the ROC curve were respectively PTX3 0. 824; BNP 0. 772; CM-KB 0. 643; CNTIO. 671, the sensitivity and specificity were PTX3 0. 8, 0. 58; CK-MB 0. 56,0. 79; CTNI 0. 60,0. 69; BNP 0. 73, 0. 58. In terms of predicting the prognosis of sepsis with heart failure complications, the PTX3 value's area under ROC curve was larger than that of BNP (respectively 0. 844, 0. 472).

CONCLUSIONThe PTX3 is an objective biochemical marker in diagnosis of sepsis; it is helpful in assessment of severity and prognosis of sepsis; it also has a certain clinical value in the assessment of sepsis cardiovascular function damage.

Adolescent ; Biomarkers ; blood ; C-Reactive Protein ; analysis ; Calcitonin ; blood ; Cardiovascular System ; physiopathology ; Child ; Child, Preschool ; Creatine Kinase ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Intensive Care Units, Pediatric ; Leukocyte Count ; Male ; Natriuretic Peptide, Brain ; blood ; Prognosis ; Protein Precursors ; blood ; ROC Curve ; Sensitivity and Specificity ; Sepsis ; diagnosis ; physiopathology ; Serum Amyloid P-Component ; analysis ; Troponin ; blood

Objective To evalute the effect of Ilizarov technique in the treatment of hypertrophic nonunion.Methods Form June 2008 to December 2010,12 patients with hypertrophic nonunion were treated with Ilizarov technique,including 10 males and 2 females with an average age of 46.5 years.The pathology sites of nonunion were kept as closed as possible without any bone graft during operation.As to patients who had ever been treated with plate or intramedullary nail,the hardware should be removed by minimal invasive approach.These procedures aimed to keep the vascularity of nonunion site intact.Ilizarov apparatus were preoperatively constructed.Distal segment and proximal segment of nonunion were mounted respectively with two external circle using the smooth wires and half pins.The two-circle stabilizing one segment was nominated with transosseous modules.Distal module and proximal one was connected with a pair of axial hinges.The pathology sites were gradually distracted from the seventh day postoperatively,0.25 mm/d.Accompanying with deformity correction,limb length discrepancy (LLD) also were restored simultaneously.Then,all the screws and nuts in the apparatus should be tightened,which was favourable to the callus consolidation.Results All 12 cases of nonunion healed without any bone graft.The fixator wearing time lasted 6-12 months,with an average of 8 months.Correction of deformity and LLD were achieved.The average lengthening was 3.0 cm (range,2.0-5.5 cm),the average correction angle was 23° (range,10°-30°).After 6-18 months follow-up,all the patients restored satisfactory function.Conclusion Hypertrophic nonunion can be treated successfully with Ilizarov technique.The key of successful callus distraction is strictly identifying the indications.

Objective To discuss the donor site complications and the treatments of the anterolateral thiIgh flap transfer. Methods From July 1988 to July 2007,427 patients treated by anterolateral thish flap surgery were followed up.The donor sites were treated by several methods,including direct closure(49 cases),skin graft transfer(258 cases),anterolateral fascial flap transfer(27 cases),musculocutaneous flap and skin sraft transfer(65 cases), superficial epigastric artery flap transfer(28cases).The area of these flaps ranged from 30 cm×10 cm to 18 cm×7 cm.and the average follow-up was 21 months(ranged from 8 months to 54 months). All the data was analyzed by SPSS for Windows,Version 11.0.1. Results Short-term complications(within 4 weeks)and long-term complications (over 6months)were observed.The former included skin necrosis(5.4%),wound infection(4.0%),and necrosis of rectus femoris(0.5%); and the latter included chronic ulcer(4.9%), aerious scar(6.6%), serious discomfortable(5.9%),and quadriceps femoris disfunction(3.7%).The statistical results showed that there was a close correlation between the donor site morbidity and the surgical methods. Conclusion The complications of the donor site cannot be ignored.Protect the soft-tissue of the donor site carefully,design all appropriate operation plan.Careful preoperative examination and local flap transplantation can effectively decrease the rate of complications.