Abstract: Objective　To analyze the epidemiological characteristics of 151 cases of melioidosis and the drug resistance of Burkholderia pseudomallei (BP), in order to provide the basis for diagnosis, treatment and reasonable prevention of melioidosis. Methods　A total of 151 inpatients and outpatients from the Second Affiliated Hospital of Hainan Medical University from January 1, 2013 to August 31, 2022 were collected, and clinical specimens were submitted for examination to isolate and identify BP strains. The clinical data of 151cases of melioidosis and the drug resistance characteristics of pathogenic bacteria were retrospectively analyzed, and using SPSS26.0 software for statistical analysis. Results　Among 151 cases with BP infection, there were 138 males (91.4%) and 13 females (8.6%); the most patients were aged from 45-<60 years old, accounting for 74 cases (49.0%); melioidosis incidence was concentrated in October (19.2%), November (19.2%), August (9.9%) and July (8.6%), and; the number of confirmed cases showed an increasing trend and the time for confirmation was <10 d; Internal medicine system (31.1%), surgery system (26.5%) and intensive care department (20.5%) were the common departments for treating melioidosis; blood (49.0%), sputum (9.9%) and wound secretion (8.6%) were the main clinical specimens for detecting BP; pulmonary infection (68.2%), sepsis (35.1%) and local suppurative infection (23.8%) were the top clinical manifestations in patients with BP infection; the effective rate of treating melioidosis was 74.8%; abnormal liver function was a risk factor for the curative effect of melioidosis (χ2=5.010, P<0.05); the sensitivity rates of BP strains to sulfamethoxazole-trimethoprim (SXT), doxycycline (DOX), imipenem(IPM), ceftazidime (CAZ), amoxicillin/clavulanate (AMC) and tetracycline (TCY) were generally more than 90%, with sensitivities of 98.7%, 97.2%, 96.7%, 94.0%, 93.2% and 90.7%, respectively. Conclusions　It can be concluded that misdiagnosis or missed diagnosis of melioidosis is easy to occur, and the understanding of the epidemiological characteristics and risk factors in this area should be strengthened. The sensitivity of BP to commonly used antibiotics has shown a certain downward trend, clinical use should be standardized, and drug resistance monitoring should be strengthened to improve the efficacy of melioidosis treatment.

OBJECTIVETo prepare a decellularized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and construct decellularized laryngeal muscles.

METHODSPerfusion decellularized larynxes were obtained by common carotid arterious perfusion with detergents. Then they were performed by macroscopic view, histological examination, scanning electron microscopy (SEM) and cartilage viability. Decellularized laryngeal scaffold were then reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day's adherence and harvested after eight weeks. Macroscopic view, histological examination and immunohistochemistry were performed.

RESULTSPerfusion larynxes became transparent after two hours. Histology and SEM indicated that perfusion method showed better decullularized effect. More vintages and collagen fibers but no intact cell or nuclei were retained in the decellularized matrix. Porosity measured by Image pro plus 6.0 was 80.4% +/- 3.2% (x +/- s). Chondrocyte vitality assay indicated chondrocyte vitality rate in the perfusion group was 86.9% +/- 1.5%. After eight weeks, vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistochemical examination indicated that sarcomeric-alpha actin expressed positively in corresponding areas.

CONCLUSIONSIt is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing tissue-engineered laryngeal muscles. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.

Animals ; Extracellular Matrix ; Feasibility Studies ; Laryngeal Muscles ; cytology ; physiology ; Larynx, Artificial ; Rabbits ; Regeneration ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo evaluate the immunogenicity of the decellularized laryngeal scaffold.

METHODSTwelve perfused, decellularized laryngeal scaffolds were obtained from rabbits through common carotid artery perfusion with detergents. The twelve decellularized laryngeal scaffolds and the twelve fresh larynxes were then implanted in para-laryngeal muscles of rabbits and harvested after two weeks, four weeks, twelve weeks and twenty-four weeks, respectively. Macroscopic view, histological examination and lymphocyte infiltration test were performed.

RESULTSThe decellularized larynxes were implanted and preserved the laryngeal extracellular matrix and laryngeal architecture. The decellularized larynx did not show obvious immunological rejection after implanted into the para-laryngeal muscles of the recipient rabbits. The volume of implanted larynx became smaller but retained cartilage scaffold. The larynxes in the control group presented the serious immunological rejection and the majority tissues of the larynxes were disintegrated and substituted by the fibrous connective tissues after four weeks. The peripheral tissues were damaged and necrotic at different degrees. The quantity of the lymphocyte infiltration in the control group was higher than that in the experiment group and the result had the statistical significance (P < 0.01).

CONCLUSIONSPerfused, decellularized technique can construct a low immune laryngeal cartilage scaffold which could be a satisfactory material for laryngeal repair.

Animals ; Cartilage ; cytology ; Cells, Cultured ; Female ; Graft Rejection ; immunology ; Larynx, Artificial ; Lymphocytes ; immunology ; Male ; Prosthesis Implantation ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds

To study the dynamic change law of bioactive constituents from Polygonum multiflorum, and to explore the optimal harvest period of P. multiflorum. Determination of stilhene glucoside, anthraquinones and catechin from P. multiflorum in different harvest times by MEKC-DAD, and principal component analysis (PCA) was used to comprehensive evaluation for bioactive constituents. There are obvious differences among the contents of active ingredients in various collecting periods samples, the content of stilbene glucoside was the highest in November, the total content of combined anthraquinone was the highest in November and December, the content of catechin was the highest in September. The comprehensive evaluation index obtained with principal component analysis showed that the sample collected in November is significantly higher than those with other samples. The optimal harvest period of P. multiflorum is November.
Electrophoresis ; Fallopia multiflora ; chemistry ; growth & development ; metabolism ; Time Factors

The aim of this study is to investigate the therapeutic effect of RegIII-proinsulin-pBudCE4.1 plasmid on streptozotocin (STZ)-induced type 1 diabetes mellitus and its underlying mechanisms. The model of type 1 diabetes mellitus was established by intraperitoneal injections of STZ (40 mg kg(-1)) to Balb/c mice for five consecutive days. Then, ten type 1 diabetic mice were intramuscularly injected with 100 microg RegIII-proinsulin-pBudCE4.1 plasmid for 4 weeks (one time/week) and the blood glucose levels were monitored every week; whereas another ten diabetic mice served as negative control group were injected with pBudCE4.1 vector at the same dose. Normal control and model control mice were treated with normal saline at identical volume under the same way. Western blotting, MTT assay, ELISA, HE staining and Tunel assay were applied to explore the underlying mechanisms. Results showed that RegIII-proinsulin-pBudCE4.1 plasmid ameliorated the hyperglycemia symptoms in diabetic mouse remarkably. It induced an immunological tolerance state in type 1 diabetic mice by inhibiting the proliferation of splenic lymphocytes and recovering Th1/Th2 balance evidenced by MTT and ELISA analysis. Furthermore, it elevated insulin concentration in the serum of type 1 diabetic mice and promoted the regeneration of beta cells supported by the results of HE staining and Tunel assay. In conclusion, RegIII-proinsulin-pBudCE4.1 plasmid possesses powerful anti-diabetic ability, which may be involved in the inducing of immunological tolerance and enhancing beta cells recovery.
Animals ; Apoptosis ; Blood Glucose ; metabolism ; Cell Proliferation ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; therapy ; Diabetes Mellitus, Type 1 ; chemically induced ; metabolism ; pathology ; therapy ; Genetic Therapy ; Hyperglycemia ; therapy ; Injections, Intramuscular ; Insulin ; blood ; Islets of Langerhans ; cytology ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Proinsulin ; genetics ; metabolism ; therapeutic use ; Proteins ; genetics ; metabolism ; therapeutic use ; Streptozocin ; T-Lymphocytes ; cytology ; Th1-Th2 Balance

Objective To explore clinical characteristics of human cytomegalovirus (HCMV) and polyomavirus (BKV and JCV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Clinical data of 53 patients with hematologic malignancies who underwent allo-HSCT from June 2016 to December 2017 were collected.HCMV, BKV and JCV loads in patients' peripheral blood and urine were monitored once a week from day 1 to day 100 after transplantation.Incidence, occurrence time, clinical manifestations, and risk factors of viral infection were analyzed.Results A total of 51 patients had viral infection, infection rate was 96.23％.HCMV, BKV, and JCV infection rates were 54.72％ (29/53), 77.36％ (41/53), and 28.30％ (15/53) respectively.Incidences of pulmonary infection, acute graft-versus-host disease (aGVHD), and hemorrhagic cystitis (HC) were 54.72％, 58.49％, and 20.75％respectively.Analysis on risk factors showed that aGVHD (OR, 24.61[95％ CI, 2.30-46.24]), pretreatment with total body irradiation (TBI) (OR, 33.39[95％ CI, 1.57-79.13]), and use of antithymocyte globulin (ATG) (OR, 24.77[95％ CI, 1.16-52.58]) were independent risk factors affecting HCMV.Human leukocyte antigen (HLA) coincidence (OR, 0.003[95％ CI, 0.00-0.10]) could reduce the risk of HCMV viruria;pretreatment with TBI (OR, 15.10[95％ CI, 1.14-39.27]) was an independent risk factor for BKV viruria, compatible blood group of donor and recipient (OR, 0.07[95％ CI, 0.01-0.64]) could reduce the risk of BKV viruria.Conclusion HCMV and polyomavirus infection in blood and urine of recipient should be monitored as soon as possible after transplantation, so as to prevent and reduce complications in time.

AIM:To investigate the mechanism of angiotensinⅡ(AngⅡ)/angiotensinⅡ type 1 receptor (AT1R)pathway activating protein phosphatase 2A(PP2A)which leads to down-regulation endothelial nitric oxide syn-thase(eNOS)phosphorylation level in mesenteric arteries of rats.METHODS: METHODS: The mesenteric arteries of adult male SD rats(weighing 160~180 g;n=90)were isolated under aseptic conditions.Firstly,to determine the effect of angiotensinⅡdown-regulated eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into normal control(control)group and AngⅡgroup.The mesenteric arteries in AngⅡgroup were incubated with AngⅡat 1×10 -7mol/L,1×10 -6mol/L and 1×10 -5mol/L for 6 h,12 h and 24 h,respectively.Secondly,to investigate the mo-lecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan(CAN; a specific AT1R blocker)+AngⅡgroup.The mesenteric arteries were pretreated with 1×10 -5mol /L CAN for 1 h,then incubated with 1×10 -7mol/L AngⅡfor 12 h in CAN+AngⅡgroup.The protein levels of eNOS,p-eNOS(Ser1177),PP2Ac,p-PP2Ac(Tyr307)and protein phosphatase 2A inhibitor 2(IPP2A2 )in the arteries were determined by Western blot.The ac-tivity of PP2A in the arteries was detected by PP2A activity kit.RESULTS:Compared with the control group,the protein level of p-eNOS(Ser1177)in the mesenteric arteries was decreased after incubated with AngⅡfor 6 h,12 h and 24 h(P<0.05).The decreasing tendency of p-eNOS(Ser1177)showed concentration-dependently,especially in 12 h and 24 h groups.The expression of eNOS protein showed no significant difference in each group.Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10-7mol/L for 12 h in vitro, the protein levels of p-eNOS(Ser1177)were down-regulated(P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS(Ser1177)(P<0.05);the protein levels of eNOS showed no significant difference in each group.Compared with the control group,the protein levels of p-PP2Ac(Tyr307)and IPP2A2 were decreased after the mesenteric arteries were trea-ted with AngⅡat 1×10 -7mol/L for 12 h(P<0.05).Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307)and IPP2A2 (P<0.05),however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group,the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡat 1× 10-7mol/L for 12 h(P<0.05).Candesarten pretreatment inhibited the activity of PP 2A significantly(P<0.05).CON-CLUSION:AngⅡincreases PP2A activity via AT1R pathway,thus leading to down-regulation eNOS(Ser1177)phospho-rylation level in mesenteric arteries.The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac(Tyr307)and IPP2A2.

Aim To assess the effects of Trillium Tschonoskii Maxim ( TTM ) decoction on Tau protein phosphorylation and synaptic development in AD model rats induced by high activity GSK-3β. Methods The SD rats were divided into five groups of ten animals, named sham-operated group ( blank group) , AD model group, TTM group (0. 5, 0. 25, 0. 125 g·kg-1 · d-1 ) . Treatment group received gavage once a day for seven days with TTM decoction, while other groups by gavage once a day for seven days with drinking water. On 2nd day by gavage, Morris water maze test was used to assess the spatial learning and memory ability of the rats. After five days' training, rats in the treat-ment groups and AD model group were injected wort-mannin ( WT, PI3K specific inhibitor ) and GF-109203X (GFX, PKC specific inhibitor) (100 μmol ·L-1 of each, total volume of 10 μL) into the right lateral ventricle. Western blot was used to detect the levels of phosphorylation Tau protein at multiple sites and the expression level of PI3K, Akt, PKC, GSK-3β(S9, T216) and synapse-associated proteins. Immu-nohistochemical method was used to detect the hyper-phosphorylation of Tau protein in hippocampus of rats. Golgi staining was applied to detect the number and morphological changes of synaptic development and dendritic spines. Nissl' s staining was employed to ob-serve the development of neonatal neurons in hippo-campus and cortex. Results Western blot showed that the phosphorylation level of Tau in hippocampus increased in model group, and the activity of GSK-3βwas up-regulated. Among them, however, in middle dose TTM group, the phosphorylation level of Tau in hippocampus decreased and the activity of GSK-3βde-creased. The expression levels of p-PKC and p-Akt in low and middle dose treatment group were higher than those in model group, thus increasing the activity of PKC and Akt to inhibit the activity of GSK-3β kinase. Immunohistochemistry also indicated that TTM could decrease the biological effects of Tau phosphorylation in hippocampus of AD rats. Western blot showed that TTM could increase the expression levels of synapsin-1 , syn-aptophysin and GluR-1 in hippocampus of AD rats. Nissl staining showed that the number of Nissl bodies in hippocampal neurons of AD model group were signif-icantly fewer than those of sham operation group, which could be increased by TTM middle and high dose group, and the complexity and dendritic spine density of hippocampal neurons in AD rats could be en-hanced as well. Conclusion TTM can effectively im-prove the cognitive function of AD rats induced by the increase of GSK-3β activity, and its possible mecha-nism may be via down-regulating the activity of GSK-3β and inhibiting the phosphorylation of tau protein and promoting the development of neurons.

PURPOSE: Little is known about combination of the circulating Epstein-Barr viral (EBV) DNA and tumor volume in prognosis of stage II nasopharyngeal carcinoma (NPC) patients in the intensity modulated radiotherapy (IMRT) era. We conducted this cohort study to evaluate the prognostic values of combining these two factors. MATERIALS AND METHODS: By Kaplan-Meier, we compare the differences of survival curves between 385 patients with different EBV DNA or tumor volume levels, or with the combination of two biomarkers mentioned above. RESULTS: Gross tumor volume of cervical lymph nodes (GTVnd, p < 0.001) and total tumor volume (GTVtotal, p < 0.001) were both closely related to pretreatment EBV DNA, while gross tumor volume of nasopharynx (GTVnx, p=0.047) was weakly related to EBV DNA. EBV DNA was significantly correlated with progress-free survival (PFS, p=0.005), locoregional-free survival (LRFS, p=0.039), and distant metastasis-free survival (DMFS, p=0.017), while GTVtotal, regardless of GTVnx and GTVnd, had a significant correlation with PFS and LRFS. The p-values of GTVtotal for PFS and LRFS were 0.008 and 0.001, respectively. According to GTVtotal and pretreatment EBV DNA level, patients were divided into a low-risk group (EBV DNA 0 copy/mL, GTVtotal < 30 cm³; EBV DNA 0 copy/mL, GTVtotal ≥ 30 cm³; or EBV DNA > 0 copy/mL, GTVtotal < 30 cm³) and a high-risk group (EBV DNA > 0 copy/mL, GTVtotal ≥ 30 cm³). When patients in the low-risk group were compared with those in the high-risk group, 3-year PFS (p=0.003), LRFS (p=0.010), and DMFS (p=0.031) rates were statistically significant. CONCLUSION: Pretreatment plasma EBV DNA and tumor volume were both closely correlated with prognosis of stage II NPC patients in the IMRT era. Combination of EBV DNA and tumor volume can refine prognosis and indicate for clinical therapy.
Biomarkers ; Cohort Studies* ; DNA* ; Herpesvirus 4, Human* ; Humans ; Lymph Nodes ; Nasopharynx ; Plasma ; Prognosis ; Radiotherapy* ; Tumor Burden*

Glioblastoma(GBM)is a lethal cancer with limited therapeutic options.Dendritic cell(DC)-based cancer vaccines provide a promising approach for GBM treatment.Clinical studies suggest that other immu-notherapeutic agents may be combined with DC vaccines to further enhance antitumor activity.Here,we report a GBM case with combination immunotherapy consisting of DC vaccines,anti-programmed death-1(anti-PD-1)and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy,and the patient remained disease-free for 69 months.The patient received DC vaccines loaded with multiple forms of tumor antigens,including mRNA-tumor associated antigens(TAA),mRNA-neoantigens,and hypochlorous acid(HOCl)-oxidized tumor lysates.Furthermore,mRNA-TAAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histo-compatibility complex(MHC)class Ⅰ and Ⅱ antigen presentation.The treatment consisted of 42 DC cancer vaccine infusions,26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions.The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells.No immunotherapy-related adverse events were observed during the treatment.Robust antitumor CD4+and CD8+T-cell responses were detected.The patient remains free of disease progression.This is the first case report on the combination of the above three agents to treat glioblastoma patients.Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient.A large-scale trial to validate these findings is warranted.