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.

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.