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Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
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Animales , Ratones , Anticuerpos , Nicotiana/genética , Agrobacterium/genética , Reactores Biológicos , Western BlottingRESUMEN
In this paper, the main objective was to raise chickens’ antibodies against three crucial public health microorganisms: the human immunodeficiency virus-1, Salmonella spp, and Staphylococcus aureus. Immunogens were prepared from the said microorganisms. Chickens were vaccinated either orally or intramuscularly. After a booster immunization, mostly eggs were collected and assess for the presence of specific antibodies. The most important results were the production of a large amount of anti-HIV antibodies in chicken’s eggs, and also the synthesis of anti-protein a antibodies with the ability to inhibit the growth of S. aureus in vitro and to serve as anti-anti-idiotypic antibodies with the capacity of neutralizing the original antigen. Enzyme- linked immune absorbent assays detected the presence of these antibodies as anti-Salmonella antibodies that were critical in reducing the bacterial load in the stomach and caeca compared with a control group. The vaccines were effective and safe, but more laboratory work, and economics have to be carried out to start a human trial.
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ABSTRACT Schirmer strips and conjunctival swabs are used in ophthalmology for the collection of tears and fluids. One of the biggest challenges during the COVID-19 pandemic has been accurate diagnosis and, in some cases, ocular manifestations are among the first symptoms. In this context, this study aimed to collect evidence to support the use of Schirmer strips and conjunctival swabs as a method of sample collection for viral analysis. A literature search was conducted following the Scoping Review protocol defined by The Joanna Briggs Institute. Studies were analyzed regarding virus research, collection methods, and sample analysis. The findings support that viruses can be detected on the ocular surface through analysis of Schirmer strips and conjunctival swabs. However, additional studies with larger samples and time data are necessary to confirm these conclusions.
RESUMO A fita de Schirmer e o swab conjunctival são utilizados na oftalmologia como métodos de coleta para lágrimas e fluidos. Durante a pandemia da COVID-19, um dos desafios foi o diagnóstico correto e se sabe que, em alguns casos, as manifestações oculares podem ser um dos primeiros sintomas. Nesse contexto, este estudo tem como objetivo levantar evidência que destaque o uso de fitas de Schirmer e de swabs conjuntivais como método de coleta para análise viral. Conduziu-se uma revisão de literatura seguindo o protocolo para Scoping Review definido pelo Joanna Briggs Institute. Os pesquisadores analisaram os estudos em busca do vírus pesquisado, os métodos de coleta e os métodos de análise. Vírus podem ser detectados na superfície ocular através da análise de fitas de Schirmer e de swabs conjuntivais, entretanto novos estudos com populações maiores e com definições claras de tempo são necessários para conclusões mais assertivas no tema.
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ABSTRACT Staphylococcus hominis (S. hominis) is a coagulase-negative Staphylococci and an infrequent cause of endophthalmitis. Due to its ability to produce biofilm, especially in diabetic patients, strains may acquire antibiotic resistance. We present two cases of S. hominis endophthalmitis, one with acute endophthalmitis after intravitreal bevacizumab injection and one with chronic endophthalmitis following undiagnosed penetrating ocular trauma. Although there are only four published S. hominis endophthalmitis cases in the literature, to the best of our knowledge, there has been no previously published case after intravitreal bevacizumab.
RESUMO Staphylococcus hominis (S. hominis) é um estafilococo coagulase-negativo e uma causa pouco frequente de endoftalmite. Devido à sua capacidade de produzir biofilme, especialmente em pacientes diabéticos, cepas dessa bactéria podem adquirir resistência a antibióticos. Este relato apresenta dois casos de endoftalmite por S. hominis: um de endoftalmite aguda após injeção intravítrea de bevacizumabe e outro de endoftalmite crônica após trauma ocular penetrante não diagnosticado. Embora existam apenas quatro casos de endoftalmite por S. hominis publicados na literatura, até onde sabemos não houve nenhum caso publicado anteriormente após bevacizumabe intravítreo.
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Resumen La uroporfirinógeno descarboxilasa humana (UROD-h) es la quinta enzima del camino biosintético del hemo y su actividad deficiente, relacionada a mutaciones en su gen, se encuentra asociada a un subgrupo de porfirias. El objetivo de este trabajo fue estudiar la relación entre la dimerización de la enzima y su actividad enzimática y comprobar si la dimerización de UROD-h es imprescindible tanto para la primera etapa de la reacción (urogen→heptagen), como para la segunda etapa (heptagen→coprogen). Con ese objetivo, se expresó y purificó la UROD-h hasta homogeneidad, se analizó el comportamiento dímero-monómero bajo distintas condiciones que pudieran desplazar el equilibrio de dimerización y se evaluó la actividad enzimática en dichas condiciones. Los resultados obtenidos sugieren que la especie activa para la primera etapa de la reacción es el homodímero y que tanto el dímero como el monómero se comportan como especies activas para la segunda etapa de la reacción. Se propone que mutaciones clínicas como la Y311C, existentes en pacientes con porfiria cutánea tarda, podrían afectar la estabilidad del dímero y podrían ser el blanco para futuras terapias génicas.
Abstract Human uroporphyrinogen decarboxylase (UROD-h) is the fifth enzyme in the heme biosynthetic pathway and its deficient activity, related to mutations in its gene, is associated with a subset of porphyrias. The objective of this work was to study the relationship between the dimerisation of the enzyme and its enzymatic activity and to verify if the dimerisation of UROD-h is essential both for the first stage of the reaction (urogen→heptagen), and for the second stage (heptagen→ coprogen). With this objective, the UROD-h was expressed and purified to homogeneity, the dimer- monomer behaviour was analysed under different conditions, which could shift the dimerisation equilibrium, and the enzymatic activity was evaluated under these conditions. The results obtained suggest that the active species for the first stage of the reaction is the homodimer, and both the dimer and the monomer behaved as active species for the second stage of the reaction. It is proposed that clinical mutations such as Y311C, existing in porphyria cutanea tarda patients, could affect dimer stability and could be the target of future gene therapies.
Resumo A enzima uroporfirinogênio descarboxilase humana (UROD-h) é a quinta enzima da via biossintética do heme e sua atividade deficiente, relacionada com mutações em seu gene, está associada a um subgrupo de porfirias. O objetivo deste trabalho foi estudar a relação entre a dimerização da enzima e sua atividade enzimática e comprovar se a dimerização da UROD-h é imprescindível tanto para a primeira etapa da reação (urogênio→heptagênio), quanto para a segunda etapa (heptagênio→coprogênio). Com esse objetivo, a UROD-h foi expressa e purificada até a homogeneidade, o comportamento de dímero-monômero foi analisado sob diversas condições, que puderam deslocar o equilíbrio de dimerização, e a atividade enzimática foi avaliada em tais condições. Os resultados obtidos sugerem que a espécie ativa para a primeira etapa da reação é o homodímero, e tanto o dímero quanto o monômero se comportam como espécies ativas para a segunda etapa da reação. Propõe-se que mutações clínicas como Y311C, existentes em pacientes com porfiria cutânea tardia, poderiam afetar a estabilidade do dímero e poderiam ser o alvo de futuras terapias gênicas em porfiria cutânea tardia.
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ObjectiveTo analyze the clinical features， treatment and prognosis of patients with respiratory depression caused by glufosinate poisoning. MethodsThe clinical data of patients with respiratory depression caused by glufosinate poisoning admitted to the ICU of Xiangshan first people’s hospital medical and health group from March 2018 to January 2022 were retrospectively analyzed. ResultsA total of 21 patients with respiratory depression caused by glufosinate poisoning were included. The median （interquartile） intake of glufosinate was 30 （20， 40） g， and the median （interquartile） visit time was within 2.0 （1.0， 2.8） h. The initial symptoms were nausea and vomiting in 16 cases （76.2%）， and sore throat in 8 cases （38.1%）. Respiratory depression， convulsions and shock occurred 6‒48 hours after ingestion of glufosinate. Convulsion occurred in 13 cases （61.9%）， shock in 10 cases （47.6%） and bradycardia in 5 cases （23.8 %）. Among the patients with convulsion or shock， respiratory depression occurred earlier than convulsion and shock in 10 cases （76.9%） and 9 cases （90.0%）， respectively. All patients were treated with gastric lavage， catharsis， mechanical ventilation and symptomatic support. Blood purification was performed in 14 cases. The duration of mechanical ventilation was 5.0 （4.0， 7.0） d，and no patient died. The patients were divided into blood purification group and routine treatment group. There was no significant difference in complications and duration of mechanical ventilation between the blood purification group and the routine treatment group （P>0.05）. ConclusionRespiratory depression caused by glufosinate poisoning usually occurs earlier than convulsion and shock. The overall prognosis of patients with respiratory depression caused by glufosinate poisoning is good， which mainly depends on the early recognition and intervention of respiratory depression.
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@#Objective To express CamA(Clostridiodies difficile adenine methltransferase A,CamA)protein with methylase activity in prokaryotic expression system.Methods The gene sequence of CamA protein of the standard strain of C.difficile1870 was amplified by PCR,cloned into plasmid pGEX-4T-1-MBP,transformed into E.coli HB101 and induced by 1 mmol/L IPTG to express the recombinant target protein. After purification with Amylose Resin,CamA protein was digested by tobacco etch virus(TEV)protease,and verified for its methylase activity using MTase-Glo~(TM)Methyltransferase Assay in vitro.Results PCR sequencing showed that the cloned CamA gene sequence was correct,in which no base mutation occurred. The recombinant expression plasmid pGEX-4T-1-MBP-CamA was digested by EcoRⅠand BamHⅠ,which showed that 1 731 bp of CamA gene was connected to the vector plasmid. The relative molecular mass of the recombinant protein was about 108 800(with 1 MBP tag),and the purity was over 90% after purified with Amylose Resin. Under the condition of20 μmol/L DNA and 20 μmol/L SAM at room temperature for 30 min,0. 5 μg CamA produced SAH at a concentration of about 101. 60 nmol/L and the enzyme activity was(0. 339 ± 0. 027)U/mg.Conclusion The CamA protein of C.difficile has been successfully expressed and purified with methylase activity,which lays a foundation of further study on the function of CamA and its role in the occurrence,development and treatment of C.difficile infection.
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Wasp sting is a common emergency in mountainous areas of China, with rapid onset and progression, high mortality rate, and serious harm to public health. Wasp sting can cause mild local reactions in mild cases, and Anaphylaxis or even multiple organ dysfunction in severe cases, of which Acute kidney injury (AKI) is the most common and serious. Blood purification treatment is commonly used for wasp sting patients to maintain renal function, eliminate toxins, and maintain Internal environment stability. The commonly used clinical methods are Hemoperfusion (HP), plasma exchange (PE), and continuous renal replacement therapy (CRRT). At present, there is no clear recommendation for the blood purification treatment mode of wasp sting in China, and there is no clear guidance for its combined treatment mode. This article will review the single and combined use of blood purification treatment models for wasp stings, based on the latest clinical research.
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Severe acute pancreatitis (SAP) is a common gastrointestinal disease, often accompanied by systemic inflammatory reactions and organ dysfunction. SAP has an acute onset, severe condition, rapid progression, and poor prognosis. The development of SAP is closely related to the excessive release of inflammatory factors. In the comprehensive treatment of SAP, continuous blood purification (CBP) can clear inflammatory mediators, improve the stability of Internal environment, improve organ function, reduce blood lipids, regulate immunity, and significantly improve the condition of SAP patients. It is an important means of treating SAP. This article reviews the research progress of CBP in the treatment of SAP.
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Objective:To compare the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification (CBP), and to provide guidance for CBP anticoagulation in children with septic shock.Methods:A case control study was conducted. Thirty-seven children with septic shock admitted to the pediatric intensive care unit (PICU) of the First Affiliated Hospital of Gannan Medical University from July 2019 to September 2022 were enrolled as the research subjects. The patients were divided into citrate local anticoagulation group and heparin systemic anticoagulation group according to different anticoagulation methods. The baseline data, the level of coagulation indicators [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), D-dimer] before treatment and 1 day after weaning from CBP, serum inflammatory mediators [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), hypersensitivity C-reactive protein (hs-CRP), procalcitonin (PCT)], bleeding complications during CBP and 7-day mortality were collected.Results:A total of 37 cases were enrolled finally, including 17 cases with citric acid local anticoagulation and 20 cases with heparin systemic anticoagulation. There was no statistically significant difference in general data such as gender, age, and body weight of children between the two groups. There were no statistically significant differences in baseline levels of coagulation indicators and inflammatory mediators before treatment of children between the two groups. One day after weaning from CBP, both groups showed varying degrees of improvement in coagulation indicators compared with those before treatment. Compared with before treatment, the PT of the heparin systemic anticoagulation group was significantly shortened after 1 day of weaning (s: 11.82±2.05 vs. 13.64±2.54), APTT and TT were significantly prolonged [APTT (s): 51.54±12.69 vs. 35.53±10.79, TT (s): 21.95±4.74 vs. 19.30±3.33], D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 4.94±3.94), with statistically significant differences (all P < 0.05). While in the citrate local anticoagulation group, only APTT was significantly prolonged after treatment compared with that before treatment (s: 49.28±10.32 vs. 34.34±10.32, P < 0.05). There were no statistically significant differences in other coagulation indicators compared with before treatment. Compared with the citric acid local anticoagulation group, the PT of the heparin systemic anticoagulation group was significantly shortened after treatment (s: 11.82±2.05 vs. 13.61±3.05, P < 0.05), and the D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 3.77±2.38, P < 0.01). The levels of inflammatory mediators in both groups were significantly reduced 1 day after CBP weaning compared with those before treatment [citric acid local anticoagulation group: hs-CRP (mg/L) was 12.53±5.44 vs. 22.65±7.27, PCT (μg/L) was 1.86±1.20 vs. 3.30±2.34, IL-6 (ng/L) was 148.48±34.83 vs. 202.32±48.62, TNF-α (ng/L) was 21.38±7.71 vs. 55.14±15.07; heparin systemic anticoagulation group: hs-CRP (mg/L) was 11.82±4.93 vs. 21.62±8.35, PCT (μg/L) was 1.90±1.08 vs. 3.18±1.97, IL-6 (ng/L) was 143.81±33.41 vs. 194.02±46.89, TNF-α (ng/L) was 22.44±8.17 vs. 56.17±16.92, all P < 0.05]. However, there was no statistically significant difference between the two groups (all P > 0.05). There was no statistically significant difference in bleeding complication during CBP and 7-day mortality in children between the citrate local anticoagulation group and the heparin systemic anticoagulation group (5.9% vs. 30.0%, 17.6% vs. 20.0%, both P > 0.05). Conclusions:Heparin for systemic anticoagulation and regional citrate anticoagulation can significantly reduce the levels of IL-6, TNF-α, hs-CRP and PCT in children with septic shock, and relieve inflammatory storm. Compared with citric acid local anticoagulation, heparin systemic anticoagulation can shorten the PT and reduce the level of D-dimer in children with septic shock, which may benefit in the prevention and treatment of disseminated intravascular coagulation (DIC).
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Objective:To compare the effect and safety of continuous veno-venous hemofiltration (CVVH)+double plasma molecular absorption (DPMA)+hemoperfusion (HP), CVVH+HP, and CVVH+plasma exchange (PE) in treatment of patient with severe wasp stings injury.Methods:Multicenter, historical cohort study and superiority test were used. From July 2020 to October 2022, patients with wasp sting injury and multiple organ damage admitted to the intensive care units (ICU) of five hospitals were consecutively screened and recruited into the CVVH+DPMA+HP group (intervention group). Propensity score matching was used to establish historical cohorts. Patients with severe wasp sting injury who hospitalized from January 2016 to June 2020 in each ICU were collected and matched 1∶1 with the intervention group, and divided into CVVH+HP group and CVVH+PE group according to their actual hemopurification protocols (historical control groups). The primary outcome was the acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score on days 3 and 7 after initiation of treatment. Secondary outcomes included complications, length of ICU and hospital stays, and all-cause mortality. Multivariate Cox proportional risk regression was used to analyze the prognosis of patients.Results:After propensity score matching, 56 patients in intervention group and each of the two historical control groups were matched successfully. There were no significant differences in age, gender, comorbidities, biochemical test indices and critical illness scores among the groups. After treatment, APACHE Ⅱ score markedly declined in all groups, and the decrease was faster in the intervention group; treatment with DPMA [hazard ratio ( HR) = 1.04, 95% confidence interval (95% CI) was 1.02-1.08, P = 0.00], the decreased levels of body temperature ( HR = 1.02, 95% CI was 1.00-1.03, P = 0.02), serum creatine kinase (CK; HR = 0.98, 95% CI was 0.96-1.00, P = 0.05) and myoglobin (MYO; HR = 2.88, 95% CI was 1.24-6.69, P = 0.01) were independent risk factors for APACHE Ⅱ score decline to the target value (15 scores). There were no significant differences in the incidence of bleeding complications, filter or perfusion thrombosis, blood pressure reduction, catheter-related infection and anaphylaxis among the groups. Conclusion:CVVH+DPMA+HP regimen can significantly reduce the APACHE Ⅱ score of patients with severe wasp sting injury, and the efficacy is superior to CVVH+HP and CVVH+PE regimens, with safety.
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Objective:To investigate the effect of bedside high-flow continuous blood purification (CBP) combined with Xuebijing in the treatment of severe sepsis (SS) and the influence on the patient′s coagulation-fibrinolysis index, immunity index and expression of peripheral blood Toll-like receptor 4 (TLR4).Methods:Ninety-three patients with SS who were admitted and treated in the Lianyungang First People′s Hospitalfrom January 2017 to October 2019 were selected. They were divided into the combined group (51 cases, treatment with bedside high-flow CBP and Xuebijing injection based on bundle therapy) and the control group (42 cases, treatment with Xuebijing injection based on bundle therapy). The changes in coagulation and fibrinolysis index, immunity index, biochemical index such as TLR4 before treatment and after 1 week of treatment were compared between the two groups. The incidences of complications in both groups were statistically analyzed, and the discharge time from ICU, mechanical ventilation time and 28-day mortality were recorded.Results:After 1 week of treatment, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) in the two groups were shortened, D-dimer (D-D) and fibrinogen (FIB) were decreased ( P<0.05); and the levels of PT and APTT in the combined group were shorter than those in the control group, the levels of DD and FIB were lower than those in the control group, there were statistical differences ( P<0.05). After 1 week of treatment, the levels of CD 4+ and CD 4+/CD 8+ ratio in both groups were increased ( P<0.05), and the levels of CD 4+ and CD 4+/CD 8+ ratio in the combined group were higher than those in the control group ( P<0.05). After 1 week of treatment, the levels of TLR4, C-reactive protein (CRP), procalcitonin (PCT), white blood cell count (WBC), blood lactate (Lac), blood urea nitrogen (BUN) and serum creatinine (Scr) in both groups were decreased ( P<0.05), meanwhile, the above indexes in the combined group were lower than those in the control group ( P<0.05). The incidence of multiple organ failure and the 28-day mortality rate in the combined group were lower than those in the control group: 3.92%(2/51) vs. 19.05%(8/42), 13.73%(7/51) vs. 30.95%(13/42), there were statistical differences ( P<0.05). The discharge time from ICU and mechanical ventilation time in the combined group were shorter than those in the control group: (12.35 ± 2.14) d vs. (14.17 ± 3.36) d, (7.12 ± 2.23) d vs. (8.51 ± 2.39) d, there were statistical differences ( P<0.05). Conclusions:Bedside high-flow CBP combined with Xuebijing injection in the treatment of SS can improve the patient′s condition, regulate the balance of coagulation and fibrinolysis, avoide the activation of coagulation, inhibite inflammatory response, reduce the expression of TLR4 in peripheral blood, improve immune function, protecte kidney function and promotethe patient′s recovery.
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Objective:To explore the scope, mode, anticoagulation mode and complications of blood purification in children with acute and critical illness.Methods:A total of 377 times of treatment of 102 children treated with blood purification in PICU at the First Affiliated Hospital of Xinxiang Medical College from January 2018 to December 2020 were retrospectively analyzed.Results:Among 102 critically ill children treated with blood purification, acute and chronic renal failure ranked the first in terms of disease distribution, with 23 cases in total, followed by 16 cases of severe viral encephalitis (meningoencephalitis), 11 cases of septic shock, seven cases of acute poisoning, five cases of severe allergic purpura, five cases of necrotic encephalopathy.In terms of clinical prognosis, 51(50.0%) cases were cured, 29(28.4%) cases were improved, 10(9.8%) cases died, and 12 cases abandoned treatment.In 2019, the blood purification application frequency was the highest, with a total of 47 cases, which was higher than those in 2018 and 2020( P<0.05). Continuous veno-venous hemofiltration was used in the largest number of children, with a total of 56 cases.There was a statistically significant difference in the application ratio of this mode during 3 years ( P<0.05), while there was no statistically significant difference in the application ratio of other modes.In terms of the selection of anticoagulation methods, the proportions of systemic anticoagulation and extracorporeal anticoagulation had significantly difference among different years( P<0.05), and the application of extracorporeal anticoagulation had increased year by year.There was no statistically significant difference in the proportion of patients without anticoagulants.The incidence of complications of blood purification was the highest in 2019, with catheter related thrombus in the majority (30 person-times), followed by hypothermia, catheter filter coagulation, hematoma formation, catheter related infection, hypotension, heparin-induced thrombocytopenia, etc.There was statistically significant difference in the total complications among different years( P<0.05). Conclusion:Blood purification is widely used in children with acute and critical illness, with a variety of diseases.The most commonly used mode is continuous veno-venous hemofiltration and in vitro anticoagulation.Catheter-related thrombosis is the most common complication.
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Objective:To compare the characteristics of patients undergoing blood purification treatment in PICU of a children′s tertiary hospital during 8 years, so as to analyze the changes in the development of blood purification technology in children in East China.Methods:Patients who received blood purification treatment in PICU of Children′s Hospital of Fudan University from 2014 to 2021 were included and divided into two study periods: 2014-2017 and 2018-2021.The clinical characteristics and treatment parameters of patients were collected and analyzed.Results:A total of 1 029 patients were included in the study, of which 103 were combined with extracorporeal membrane oxygenation.The 28-day survival rate of 926 patients treated with pure blood purification was 55.7%.Among them, patients with younger age, lower body weight, using mechanical ventilation, using vasoactive drugs before blood purification, and patients with multiple organ dysfunction syndrome had a higher distribution in the death group than those in survival group( P<0.05). During 8 years, a total of 3 688 cases of blood purification were performed.The main mode was continuous veno-venous hemodiafiltration (CVVHDF) (68.6%), followed by therapeutic plasma exchange (TPE) (23.8%) and hemoperfusion (HP) (4.8%); the main indication was acute kidney injury (AKI) (29.3%), followed by severe inflammatory disease (26.2%) and acute liver failure (16.2%). Compared with 2014-2017, the number of blood purification treatments in 2018-2021 increased by 47.4%, and the survival rate of patients increased significantly (48.7% vs. 58.1%, P<0.05). The distribution of blood purification patterns and indications also changed( P<0.05). The proportions of TPE (20.5% vs. 26.0%) and HP (3.1% vs. 6.0%) increased, while the proportion of CVVHDF (71.9% vs. 66.4%) decreased significantly.The proportions of AKI (29.8% vs. 38.9%) and refractory immune diseases (8.4% vs. 15.2%) were significantly higher, while severe inflammatory diseases (29.2% vs. 24.2%) and acute liver failure (19.6% vs. 13.8%) had declined. Conclusion:From 2014 to 2021, the number of blood purifications performed in our center increased significantly.Although the distribution of indications and patterns have also changed significantly, the overall survival rate is significantly improved.However, standardized practice still needs to be strengthened.
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The temporomandibular joint (TMJ) is surrounded by a joint capsule, the smooth inner layer of which is called the synovial membrane. Synovium is involved in various intraarticular diseases, and it is a key area of joint disease. Synovial type-A cells are located in the lining layer of the synovial membrane, mostly on the side of the membrane close to the joint cavity. They have a strong phagocytic effect, and their main role is to remove the degradation products of the intra-articular and extracellular matrix. Various intra-articular diseases will affect the synovium, which is the key area of joint disease. The method of cell culture in vitro can effectively simulate the growth environment of cells in vivo and can accurately understand the effects of single and multiple factors on synovial cells, which has become a basic research method. In this review paper, the latest research progress in human temporomandibular joint type-A synoviocytes is reviewed from the aspects of cell origin, in vitro culture, cell purification, and cell biological function.
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@#ObjectiveTo explore the purification effect of hepatitis A virus(HAV)with a new type of composite medium Capto Core 400 chromatography packing.MethodsHAV crude liquid cultured with human embryonic lung diploid cells(2BS strain)was purified using Capto Core 400 chromatography media. The optimum conditions for purification of HAV were determined from flow rates(60,150,300 and 450 cm/h),pH of loading buffer(6. 6,7. 0 and 7. 4),salt ion concentration of loading buffer(0,0. 15,0. 3 and 0. 45 mol/L)and capacity[0. 5,1,2 and 4 column volume(CV)],respectively. The purification effect was compared with that of traditional Sepharose 4FF chromatography media.Results The optimum purification conditions of Capto Core 400 were preliminarily determined as follows:flow rate of 150 cm/h,loading buffer pH of 7. 0 ～ 7. 4,salt ion concentration of 0. 15 mol/L,loading volume of 2 CV. The average recovery rate of virus was 92. 61%,and the average removal rate of protein was 84. 25%. The average recovery rate of HAV antigen purified by Sepharose 4FF chromatography media was 76. 03% and the average protein removal rate was 73. 21%. The purification effect of Capto Core 400 chromatography media was better than that of Sepharose 4FF chromatography media.ConclusionThe Capto Core 400 composite chromatography media had a high recovery rate for HAV purification and a relatively simple operation,which was suitable for HAV purification.
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@#ObjectiveTo optimize the condition for anion exchange chromatography in purification process of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein by Design of Experiment(DOE)so as to decrease the content of host cell protein(HCP)in bulk of protein.MethodsA complete factorial experimental design with four factors at two levels was established by the DOE in Minitab 19 software.The condition(flow rate,sample load,pH value of buffer and salt concentration of eluent)for anion exchange chromatography in purification process of rhGH-Fc was optimized by DOE to find out the operating space.ResultsThe experimental results were predicted accurately by the established DOE model.The sample load,pH value of buffer,salt concentration of eluent as well as the interaction of pH value of buffer and salt concentration of eluent showed significant influence on the HCP content in the harvest.The optimal sample load,flow rate as well as pH value and salt concentration of eluent were 15 mg/mL,120 cm/h,7.0 ～ 8.0 and 0.1 mol/L respectively.The HCP contents in eluents under the optimal condition were less than 400 ng/mg,which met the requirements for verification within the range of results in determined operating space.ConclusionThe condition for removal of HCP by anion exchange chromatography was successfully optimized by DOE,which provided a reference for further application of DOE in the biopharmaceutical field.
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@#ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.
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OBJECTIVE@#To analyze the clinical characteristics of hemophagocytic syndrome (HLH) children with different EB virus (EBV) DNA loads, and to explore the relationship between differential indicators and prognosis.@*METHODS@#Clinical data of 73 children with HLH treated in our hospital from January 2015 to April 2022 were collected. According to EBV DNA loads, the children were divided into negative group (≤5×102 copies/ml), low load group (>5×102-<5×105 copies/ml) and high load group (≥5×105copies/ml）. The clinical symptoms and laboratory indexes of the three groups were compared, and the ROC curve was used to determine the best cut-off value of the different indexes. Cox regression model was used to analyze the independent risk factors affecting the prognosis of children, and to analyze the survival of children in each group.@*RESULTS@#The proportion of female children, the swelling rate of liver and spleen lymph nodes and the involvement rate of blood, liver, circulation and central nervous system in the high load group were higher than those in the negative group. The incidence of disseminated intravascular coagulation(DIC) and central nervous system(CNS) involvement in the high load group were higher than those in the low load group. The liver swelling rate and circulatory system involvement rate in the low load group were higher than those in the negative group(P<0.05). PLT counts in the high load group were significantly lower than those in the negative group, and the levels of GGT, TBIL, CK-MB, LDH, TG, SF, and organ involvement were significantly higher than those in the negative group. The levels of CK, LDH, SF and the number of organ involvement in the high load group were significantly higher than those in the low load group. The levels of GGT and TBIL in low load group were significantly higher than those in negative group. In terms of treatment, the proportion of blood purification therapy in the high and low load group was significantly higher than that in the negative group(P<0.01). ROC curve analysis showed that the best cut-off values of PLT, LDH, TG and SF were 49.5, 1139, 3.12 and 1812, respectively. The appellate laboratory indicators were dichotomized according to the cut-off value, and the differential clinical symptoms were included in the Cox regression model. Univariate analysis showed that LDH>1139 U/L, SF>1812 μg/L, dysfunction of central nervous system, number of organ damage, DIC and no blood purification therapy were the risk factors affecting the prognosis of children (P<0.05); Multivariate analysis shows that PLT≤49.5×109/L and dysfunction of central nervous system were risk factors affecting the prognosis of children (P<0.05). Survival analysis showed that there was no significant difference in the survival rate among the three groups.@*CONCLUSION@#The incidence of adverse prognostic factors in children with HLH in the EBV-DNA high load group is higher, and there is no significant difference in the survival rate of the three groups after blood purification therapy. Therefore, early identification and application of blood purification therapy is of great significance for children with HLH in the high load group.
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Humanos , Niño , Femenino , Linfohistiocitosis Hemofagocítica , Estudios Retrospectivos , Factores de Riesgo , ADN , PronósticoRESUMEN
The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.