Objective To construct 6 kinds of human soluble proliferation-inducing ligand (sAPRIL) cDNA mutants. Methods Six pair primers according to the cDNA sequence of human sAPRIL for different mutants were designed as follows: HEL Th epitope was added at C or N end, the last or first 45 bp DNA was deleted and HEL Th epitope was added, the last or first 90 bp DNA was deleted and HEL Th epitope was added. Different enzyme site was designed at 5′ end of 12 primers, BamH Ⅰ or SalⅠ or HandⅢ. At the same time, 2 pairs of HEL Th epitope sequence were designed and synthesized, and different incision enzyme sticky end sequence was added at the 5′ or 3′ end of each epitope DNA. Mutant sequences were amplified by 6 pairs primers with sAPRIL cloning plasmid as template. The PCR fragment was digested and then ligated with the HEL Th epitope DNA that had been reannealed. The ligation fragments of the head were ligated with digested pQE80 vector fragment again, and were transformed into DH5? competent cell. Recombinant plasmids were identified by digestion and sequencing. Results Six sAPRIL cDNA mutant DNA sequences were obtained by PCR, and the expression plasmids of sAPRIL cDNA mutants including HEL Th epitope sequence were constructed successfully. The result of sequencing proved that mutations generated as designed fully. Conclusion Six sAPRIL cDNA mutant expression plasmids were constructed successfully.

Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5? system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.

Objective To compare inter-hospital transport and clinical outcome in severe acute respiratory distress syndrome(ARDS)patients whom were transported either on extracorporeal membrane oxygenation(ECMO) or on conventional ventilation,and to investigate the optimal means of inter-hospital transport. Methods Eleven patients with severe ARDS who were invalid under conventional ventilation and were transported from other hospitals to Tianjin Third Central Hospital from November 2009 to January 2014 were analyzed. Five patients were transported on ECMO(observation group)and 6 on conventional ventilation(control group). The clinical characteristics,outcomes, transportation,vital signs before and after transportation,respiratory parameters,and Murray score between two groups were compared. Results Patients in observation group were significantly older than those in control group〔years:73(46,77)vs. 34(23,46),Z=-2.293,P=0.022〕. There was no significant difference between observation group and control group in acute pathologic and chronic health evaluationⅡ(APACHEⅡ)score,Murray score,oxygenation index(PaO2/FiO2)before transportation,transit time,and transit distance〔APACHEⅡscore:36(33,39)vs. 27(23,35),Z=-1.830,P=0.067;Murray score:3.5±0.3 vs. 3.4±0.2,t=0.667,P=0.524;PaO2/FiO2(mmHg, 1 mmHg=0.133 kPa):61±14 vs. 63±14,t=-0.249,P=0.809;transit time(minutes):24(18,74)vs. 79(41, 86),Z=-1.654,P=0.098;transit distance(km):12.9(8.3,71.8)vs. 72.4(39.5,86.8),Z=-1.651,P=0.099〕. There was no significant difference between two groups in vital signs and respiratory parameters before transportation. When arrived in ECMO centre,heart rate,respiratory rate,fractional inspired oxygen,inspiratory pressure and Murray score in observation group were significantly lower than those in control group〔heart rate(beat/min):102±16 vs. 136±8, t=-4.374, P=0.002;respiratory rate(beat/min):23±3 vs. 37±2,t=-7.967,P=0.000;fractional inspired oxygen:0.40±0.05 vs. 0.96±0.09,t=-12.152,P=0.000;inspiratory pressure(cmH2O, 1 cmH2O=0.098 kPa):21±1 vs. 34±4,t=-6.887,P=0.000;Murray score:2.7±0.2 vs. 3.8±0.2,t=-8.573, P=0.000〕,but PaO2/FiO2 was higher than that of control group(mmHg:278±65 vs. 41±5 ,t=8.075,P=0.001). Four patients were survived in observation group,and one died from the shortage of oxygen induced lung injury deterioration during transportation. Three patients died in control group,which was directly associated with lung injury deterioration. Conclusion For patients with severe ARDS who need the support of ECMO,ECMO-assisted transfer is safer than conventional ventilation,but transfer should be implemented by experienced team.

We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Fusion ; genetics ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective:To analyze the possible causes of arrhythmia in patients receiving continuous renal replacement therapy (CRRT) with regional citrate anticoagulation (RCA).Methods:A retrospective cohort study was conducted. All patients underwent RCA-CRRT treatment from January 1, 2020 to October 31, 2020 in the intensive care unit (ICU) of Tianjin Third Central Hospital were enrolled. The patients were divided into arrhythmia group and non-arrhythmia group according to whether arrhythmia occurred. The gender, age, acute physiology and chronic health evaluationⅡ(APACHEⅡ) score, catheterization site, underlying diseases, electrocardiogram (ECG), electrolytes [total calcium, serum free calcium (iCa 2+), phosphorus, magnesium, potassium] and blood gas analysis (pH value, HCO 3-) of patients in the two groups were recorded. The changes of ECG were observed, the differences in electrolyte and blood gas analysis indexes between the two groups of patients at different time points (before CRRT, 24, 48, 72 hours after CRRT, and when arrhythmia occurred) were compared. Results:A total of 86 RCA-CRRT patients were enrolled, of which 12 cases (13.95%) had arrhythmia, and the remaining 74 cases (86.05%) had no arrhythmia. The average time for the occurrence of arrhythmia in the 12 patients was (44.00±16.82) hours. There was no significant ST-segment change in the ECG when the arrhythmia occurred compared with that before CRRT, the total calcium level was significantly higher than that before CRRT (mmol/L: 2.48±0.40 vs. 2.13±0.35, P < 0.05), the blood magnesium level was significantly lower than that before CRRT (mmol/L: 0.73±0.20 vs. 0.95±0.25, P < 0.05). There was no significant difference in iCa 2+, blood phosphorus, blood potassium, pH value and HCO 3- between before CRRT and when arrhythmia occurred. Over time, the total calcium levels in the two groups increased, and there was a statistical difference between the 48 hours after CRRT and before CRRT (mmol/L: 2.48±0.33 vs. 2.13±0.35 in the arrhythmia group, and 2.30±0.22 vs. 2.15±0.48 in non-arrhythmia group, both P < 0.05). The linear change trend of iCa 2+, pH value and HCO 3- was not obvious in the two groups. The blood phosphorus and blood magnesium levels in the two groups decreased. The blood potassium in the arrhythmia group decreased, however, the blood potassium level in non-arrhythmia group did not change significantly. The total calcium level in the arrhythmia group was significantly higher than that in the non-arrhythmia group at 72 hours after CRRT (mmol/L: 2.69±0.35 vs. 2.45±0.23, P < 0.05); however, there was no significant difference in serum iCa 2+, phosphorus, magnesium, potassium, pH value and HCO 3- between the two groups. Conclusion:Patients receiving RCA-CRRT were less likely to develop arrhythmia, the causes may be related to the accumulation of citric acid and electrolyte disturbances such as calcium, phosphorus, and magnesium.

Volume dynamics is a two-compartment dynamical model using hemoglobin (Hb) derived plasma diluted level as input data and urine output as input variable through consecutive repeated measurements of Hb concentration in the blood during infusion. It could be applied to evaluate and guide crystalloid fluid rehydration for patients with dehydration or hypovolemia and during anesthesia or surgery. Volume dynamics could be also used to quantificate of strains, hypovolume, and the change of fluid distribution and elimination caused by anesthesia or surgery. The factors which influence the volume resuscitation are complex, including gender, age, hemodynamic state [mean arterial pressure (MAP)], health and stress state, renal function, consciousness, surgical or anesthesia state and so on, which may affect the half-life, distribution, and volume of the fluid. This article summarizes and analyzes the pathophysiological changes of crystalloids fluid in vivo, in order to provide reference for volume management in critically ill patients.

Objective:To investigate the clinical effect of setting proportional pressure support (PPS) parameters by target tidal volume (VT) method.Methods:The study was conducted retrospectively on acute exacerbation of chronic obstructive pulmonary disease (AECOPD) patients admitted to Tianjin Third Central Hospital from January 2016 to December 2020. According to the PPS parameter setting method, the patients were divided into the airway blocking group and target VT group. The baseline characteristics, initial setting values of flow assist (FA) and volume assist (VA), respiratory system parameters, and clinical outcomes were collected and compared between the two groups.Results:Fifty-nine patients were enrolled, 29 patients in the airway blocking group, and 30 in the target VT group. There was no statistically significant difference in baseline characteristics, compliance, resistance, and initial settings of FA and VA between the two groups. Compared with the target VT group, the respiratory rate (RR), mean arterial pressure (MAP), VT, and arterial partial pressure of oxygen (PaO 2) recorded 1 hour after the initial setting of the PPS parameters in the airway block method group were significantly reduced [RR (times/minute): 21.0 (18.5, 22.5) vs. 23.0 (21.0, 25.0), MAP (mmHg, 1 mmHg = 0.133 kPa): 84.0 (79.0, 90.5) vs. 90.0 (87.0, 96.2), VT (mL): 305.24±41.07 vs. 330.87±46.84, PaO 2 (mmHg): 68.0 (66.0, 73.5) vs. 74.0 (69.8, 82.5), all P < 0.05], while arterial partial pressure of carbon dioxide (PaCO 2) and oral closure pressure (P0.1) were both increased significantly [PaCO 2 (mmHg): 41.0 (39.0, 46.0) vs. 37.5 (35.0, 42.2), P0.1 (cmH 2O, 1 cmH 2O = 0.098 kPa): 1.42±0.78 vs. 0.90±0.67, both P < 0.05]. Compared with airway blocking group, the duration of weaning, ICU stay, and hospital stay in the target VT group were significantly shorter [duration of weaning (hours): 42.0 (24.0, 70.5) vs. 64.0 (30.5, 97.5), ICU stay: 10.00±3.38 to 13.28±5.41, hospital stay (days): 12.07±3.40 vs. 15.41±5.60, all P < 0.05]. There was no statistically significant difference in the invasive mechanical ventilation time, weaning failure rate, ICU mortality and in-hospital mortality between the two groups. Conclusion:This study suggested that the target TV method has the advantages of practicality, safety, convenience, and rapid to set PPS parameters than the airway block method, which shortens the duration of weaning and ICU stay, and has a good clinical prospect.

Objective:To explore the predictive value of low-dose biphasic (inspiratory and expiratory) CT air trapping sign and semi-quantitative score in predicting abnormal blood gas parameters and progression to severe disease in COVID-19 patients.Methods:Patients with non-severe COVID-19 who were diagnosed by nucleic acid testing and hospitalized in designated hospitals in Wuxi City from January 23 to February 29, 2020 were prospectively and consecutively recruited. All patients received low-dose biphasic CT examination on admission and repeated CT examination at regular intervals during the course. On the inspiratory phase admission of the bipolar CT, the scope of the lesion was evaluated by semi-quantitative score, and the air trapping sign on bipolar CT was assessed. The differences of semi-quantitative score, the presence of the air trapping sign and other clinical factors were compared between the patients with abnormal and the normal blood gas index, as well as between the cases progressed to severe disease and cases without disease progression using the independent sample t-test or χ 2 test. The area under the curve (AUC) of receiver operating characteristic (ROC) and the comprehensive discriminant improvement index (IDI) were used to evaluate the predictive effectiveness of the semi-quantitative scores, air trapping sign, and combination of two factors in differentiating cases with abnormal and normal blood gas indexes, as well as in differentiating cases with and without disease progression to severe COVID-19 cases. Results:In total 51 non-severe COVID-19 cases were included, with 16 cases showed air trapping sign during the first biphasic CT examination on admission. During the course of the disease, there were 13 patients with abnormal blood gas index, and 9 cases displaying air trapping sign (9/13). All 7 cases with progression to severe cases showed air trapping sign (7/7). Patients with advanced age, air trapping sign and higher semi-quantitative score were found more likely to have abnormal blood gas index ( t=3.10, χ 2=9.38, t=3.34, P<0.05); patients with advanced age, underlying diseases, air trapping sign and higher semi-quantitative score were more likely to develop into severe disease ( t=2.68, χ 2=6.65, χ 2=4.25, t=4.33, P<0.05). The AUC of semi-quantitative score, air trapping sign and combination of two factors in distinguishing abnormal blood gas index from normal blood gas index was 0.803, 0.754 and 0.794 respectively. The AUC of semi-quantitative score, air trapping sign and combination of two factors in distinguishing cases with progression to severe cases from non-progression was 0.881, 0.898 and 0.932, respectively. Air trapping sign combined with semi-quantitative score significantly improved the prediction effectiveness of disease progression, compared with semi-quantitative score or air trapping sign (IDI=0.271, 0.117). Conclusion:Air trapping sign and semi-quantitative score might be used as effective indicators to predict the progression of COVID-19 cases, and the combination of these two factors might be more helpful to predict the disease progression.

ObjectiveTo observe the effect of surface sensation training on walking function of patients after anterior cruciate ligament reconstruction (ACLR). MethodsFrom January to November, 2021, 56 ACLR inpatients in Beijing Rehabilitation Hospital were randomly divided into control group (n = 28) and experimental group (n = 28). The control group received routine rehabilitation training of 45 minutes a time, while the experimental group received routine rehabilitation training of 30 minutes and surface sensation training of 15 minutes a time, for eight weeks. Their toe-out angle and affected side impulse percentage of plantar seven zones were measured before and after intervention. ResultsThe toe-out angle of both the healthy and the affected sides decreased in both groups after intervention (t > 4.615, P < 0.001), and it was less in the affected side in the experimental group (t = 2.263, P < 0.05). The impulse percentage in heel medial and heel lateral areas increased in both groups after intervention (t > 4.221, P < 0.001), and it was more in the experimental group (t > 3.651, P < 0.01); while the impulse percentage in middle foot, foot intermediate and foot lateral areas decreased in both groups (t > 3.174, P < 0.01), and it was less in foot intermediate and foot lateral areas in the experimental group (t > 2.366, P < 0.05); the impulse percentage decreased in foot medial and toe areas in the experimental group (t > 3.508, P < 0.01), but there was no significant difference between two groups (t < 1.608, P > 0.05). ConclusionSurface sensation training can further improve the foot deflection and distribution of impulse of affected side in patients after ACLR during walking, to normalize the load patterns.