Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C.albicans.For the detection of candicidal activity,after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above,neutrophils were stimulated with live C.albicans (MOI=1) for 60 min,serial dilutions of cell lysate were plated onto yeast agar,and CFU were enumerated after incubation at 37℃ for 48 h.The candicidal activity was represented as [1-(CFUblocking group/CFUbuffer)] × 100％.Results Under stimulation with FITC-C.albicans at a MOI of 10 for 30 and 60 min,positive percentage of intracellular Dectin-1-expressing neutrophils increased significantly when compared with initial level (0 min,8.32％ ; 30 min,16.82％ ; 60 min,23.88％) (versus 0 min,P＜0.01).However,positive percentage of cell-surface Dectin-1-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min (versus 0 min,P＞0.05).Upon blockage of Dectin-1,the stimulated ROS generation (R2=0.306,P＜0.01) and candicidal activity (R2=0.251,P＜0.01) of neutrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group.In addition,the intracellular Dectin-1 is recruited and co-distributed with ROS on the surface of phagocytized C.albicans as observed by confocal microscopy.Conclusion Taken together,these results demonstrated that an internalized expression pattern of human Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C.albicans which was phagocytized by human neutrophils.

Objective To compare the reactive oxygen species (ROS) expression in human neutrophils phagocytizing Sporothrix Schenckii and Candida albicans yeast cells,and to compare the fungicidal activity of human neutrophils against Sporothrix Schenckii and Candida albicans.Methods Human neutrophils were isolated from peripheral blood by using density gradient centrifugation method,and cultured with the presence of the yeast phase of a Sporothrix Schenckii clinical isolate and a standard strain of Candida albicans (ATCC 90028)at a multiplicity of infection of 10 or 1 for 60-210 minutes.Subsequently,flow cytometry with ROS probe (2',7'-dichlorofluorescein diacetate,DCFH-DA) was carried out to for the real time detection of intracellular ROS level,confocal laser scanning microscopy (CLSM) for the observation of ROS distribution.In addition,the fungicidal efficiency of neutrophils against Sporothrix Schenckii and Candida albicans was estimated by the number of colonies after additional culture of neutrophil lysates on brain-heart infusion agar (BHIA) medium.Statistical analysis was done by using univariate analysis of variance and LSD-t test.Results The intracellular ROS level peaked at 60 minutes in neutrophils incubated with Sporothrix Schenckii yeast cells,then decreased rapidly from 60 minutes to 210 minutes.Compared with the neutrophils incubated with Candida albicans yeast cells,those with Sporothrix Schenckii yeast cells showed a higher ROS level (expressed as mean fluorescence intensity) at 60minutes (159.67 ± 11.34 vs.112.22 ± 9.66,P＜ 0.01),but a lower ROS level at 120 minutes (89.01 ± 9.81 vs.110.25 ± 7.28,P＜ 0.05) and 180 minutes (57.63 ± 8.46 vs.109.98 ± 9.00,P＜ 0.01).CLSM revealed that ROS was mainly distributed in neutrophils with phagocytized fungal spores,and especially on the surface of phagocytized spores.Furthermore,the percentage of yeast cells killed by neutrophils was significantly lower for Sporothrix Schenckii than for Candida albicans at 180 minutes (19.21％ ± 3.68％ vs.26.63％ ± 4.97％,P ＜ 0.01).Conclusions Differential expression of intracellular ROS was observed in neutrophils after phagocytosis of Candida albicans and Sporothrix schenckii.Neutrophils exert a stronger fungicidal activity against Sporothrix Schenckii in comparison with Candida albicans,which may be associated with the rapid decrease of ROS level in neutrophils after phagocytosis.

Objective:To compare our self-designed elastic syndesmosis hook plate (ESHP) and suture-button technique in treatment of injury to the distal tibiofibular syndesmosis.Methods:A retrospective study was conducted of the 53 patients who had been treated at Military Orthopaedic Center, 909 Hospital of Joint Service Support Force of PLA for ankle fractures complicated with syndesmosis injury by ESHP or suture-button technique from March 2013 to March 2017. Of them, 28 were treated by ESHP (ESHP group: 15 males and 13 females aged from 26 to 60 years) and 25 by suture-button technique (suture-button group: 14 males and 11 females aged from 24 to 59 years). The 2 groups were compared in terms of syndesmosis fixation time, postoperative incision bleeding, hospital stay, and ankle dorsiflexion and plantarflexion and ankle function by Baird-Jackson scoring at 3, 6, 12 months after surgery. Postoperative complications were observed.Results:The 2 groups were comparable because there was no statistically significant deference in general data between the 2 groups ( P>0.05). Surgery went on uneventfully in all the patients. The syndesmosis fixation time [(9.7±2.2) min] and postoperative incision bleeding [(49.3±10.4) mL] in the ESHP group were significantly less than in the suture-button group [(16.2±1.4) min and (62.4±6.3) mL] ( P<0.05); the maximum plantar flexion (29.9°±1.3°) and Baird-Jackson scores (87.2±2.9) at 3 months after surgery in the ESHP group were significantly greater than in the suture-button group (22.8°±1.3° and 78.7±4.1) ( P<0.05). There were no significant differences between the 2 groups in hospital stay, maximum plantar flexion at postoperative 6 or 12 months, maximum dorsiflexion at postoperative 3, 6 or 12 months, or Baird-Jackson scores at postoperative 6 or 12 months (all P>0.05). This cohort was followed up for 12 to 14 months (average, 12.5 months). All fractures united during follow-up and all the implants were removed around postoperative 12 months. Follow-up within 12 months observed internal fixation failure caused by metal fatigue in one case in the ESHP group, and internal fixation irritation in one case, internal fixation failure in 2 cases and internal fixation sinking and osteolysis in one case in the suture-button group. Conclusion:Compared with the suture-button technique, treatment of injury to the distal tibiofibular syndesmosis with ESHP may lead to shorter fixation time, less postoperative bleeding and complications, and faster functional recovery of the ankle.

Objective:To investigate the clinical efficacy of distal tibiofibular screw fixation combined with ligament repair for the treatment of ankle fracture accompanied with distal tibiofibular syndesmosis injuries.Methods:A retrospective cohort analysis was made on clinical data of 72 patients with ankle fracture accompanied with distal tibiofibular syndesmosis injuries admitted to 909th Hospital of Joint Service Support Force of PLA from September 2017 to September 2020, including 38 males and 34 females, aged 19-65 years [(42.5±12.2)years]. The fracture was type B in 30 patients, type C 1 in 29 and type C2 in 13 according to Danis-Weber classification. A total of 36 patients were treated by distal tibiofibular screw fixation combined with ligament repair (fixation+repair group) and 24 patients by distal tibiofibular screw fixation (fixation group). Operation time, intraoperative blood loss and hospital stay were recorded. Visual analogue scale (VAS), American Orthopedic Foot and Ankle Society (AOFAS) ankle hindfoot score and ankle range of motion (ROM) were determined at 1, 3 months postoperatively and at the final follow-up. Complications were documented. Results:All patients were followed up for 12-22 months [(15.9±2.2)months]. There were no significant differences in operation time, intraoperative blood loss and hospital stay between the two groups (all P>0.05). VAS in fixation+repair group was (3.1±1.0)points and (2.1±0.6)points at 1, 3 months postoperatively, significantly lower than (3.9±0.8)points and (2.6±0.8)points in fixation group (all P<0.05), but the score showed no significant difference between the two groups at the final follow-up ( P>0.05). AOFAS score in fixation+repair group was (64.7±4.0)points, (73.2±3.4)points and (87.2±3.4)points at 1, 3 months postoperatively and at the final follow-up, significantly higher than (60.1±4.9)points, (70.2±1.9)points and (84.1±2.6)points in fixation group (all P<0.05). There was no significant difference in the ROM between the two groups at 1 month postoperatively ( P>0.05), but the ROM at 3 months postoperatively and at the final follow-up in fixation+repair group [(44.4±2.9)° and (52.3±2.5)°, respectively] was significantly higher than that in fixation group [(41.4±2.7)° and (50.1±2.7)°, respectively] (all P<0.05). There was 1 patient with internal fixation loosening in fixation+repair group, with the incidence of complications of 3% (1/36). While the incidence of complications was significantly lhigher in fixation group [17%(6/36)], including 2 patients with re-separation of distal tibiofibular syndesmosis, 2 with internal fixation loosening and 2 with internal fixation breakage ( P<0.05). Conclusion:Compared with distal tibiofibular screw fixation, distal tibiofibular screw fixation combined with ligament repair for the treatment of ankle fracture accompanied with distal tibiofibular syndesmosis injuries is effective to early relieve ankle pain, improve ankle function and ROM, and reduce occurrence of complications.