OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

ObjectiveTo analyze the drop-out rate of participants in antidepressant clinical trials and to explore the related influencing factors. MethodsA retrospective analysis was carried out on the participants of 9 antidepressant clinical trials conducted at the Affiliated Brain Hospital of Guangzhou Medical University from 2013 to 2020. A self-compiled questionnaire was used to collect the subjects' demographic data， disease characteristics and the final completion of the trial， thereafter， the participant drop-out rate and related influencing factors were discussed. ResultsA total of 157 cases were enrolled， including 120 cases completed and 37 cases dropped out the trail. The causes of drop-out were poor efficacy in 13 cases （35.14%）， presence of adverse reactions in 12 cases （32.43%）， withdrawal of informed consent in 8 cases （21.62%） and loss of follow-up in 4 cases （10.81%）. Correlation analysis showed that participant drop-out was positively correlated with the level of anxiety （r=0.224， P<0.01） and presence of adverse events （r=0.158， P<0.05）， meantime， negatively correlated with the level of education （r=-0.209， P<0.01） and overall efficacy （r=-0.545， P<0.01）. Binary Logistic regression analysis showed that education level （β=-0.611， OR=0.543， P<0.05）， number of visits （β=-1.831， OR=0.160， P<0.01） and overall efficacy （β=-2.286， OR=0.102， P<0.01） were the influencing factors of participant drop-out. ConclusionLow education level， first visit， poor outcome， high level of anxiety， and adverse events are the factors affecting participant drop-out in antidepressant clinical trials.

Objective To explore the technical advantages and clinical value of dual-source and image re-construction technique application in lumbar spondyloschisis without sliding .Methods 36 cases of patients with LSWS were collected who were examined by dual-source CT scan and diagnosed definitely .18 cases of patients were examined by X-ray( including lumbar vertebrae and double oblique ) .The diagnosis rate of X-ray and CT scanning in diagnosing LSWS were calculated ,which was made statistical analysis .The image of 36 cases patients with LSWS were reconstructed by multi-planar reconstruction ( MPR) ,volume rendering technique ( VR) and curved planar reconstruc-tion( CPR) .The display rate was calculated , which was made statistical analysis according to various image recon-struction in diagnosing LSWS .Results 7 cases of patients with LSWS were found by X-ray examination .36 cases of patients with LSWS were found by dual-source CT examination .The display rate was 38.9%and 100.0%.71 LSWS were found in 36 cases of patients with LSWS ,35 cases were bilateral spondylolysis ,1 case was unilateral spondylolys-is.In several image reconstruction methods ,CPR and cutting VR showed the highest rate in diagnosing LSWS ,which was 100.0%.The symptom of LSWS in X-ray examination:local thinning and structural disorder in lumbar spondylol-ysis,cortical discontinuity .The symptom of LSWS in CT examination were as follows:clear linear low density shadow in lumbar,ends hardening and bone fragments in some case .Conclusion The dual-source CT and its image recon-struction technology have the technological superiority and higher clinical value in diagnosing LSWS ,which is crucial to prevent LSWS misdiagnosed ,and could become the preferred examination in screening and diagnosing LSWS .

Objective To report the operative technique and clinical experiences of the modified reversed superficial peroneal neurocutaneous island flaps for reconstruction of the ankle and foot.Methods According to the previous anatomical studies and our clinical experiences,we devised the reversed superficial peroneal neuroeutaneous island flap based on the descending branch of the distal perforator of the peronealrtery and its venae comitantes,and covered the soft defect of the ankle and foot with it.Results Twenty-one of the 23 flaps survived completely without complications,while the other two occurred marginal necrosis.The maximum surface of the flap in our series was 12 cm×13 cm.and the minimum one was 5 cm×4 cm.The length of the pedicle ranged from 5 cm to 10 cm.The texture of the flaps was good,while the cosmetic and function of them were evaluated as acceptable in all cased after 6 to 21 months follow-up.Conclusion The reversod superficial peroneal neurocutaneous island flaps is a versatile,reliable procedure useful in reconstruction of the ankle and foot.