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 explore the potential facilitators and barriers to healthy behavior among stroke patients.Methods:Semi-structured interviews were conducted among 16 stroke patients from September 2022 to March 2023 using an objective sampling method.The interview guide was developed using the theoretical domain framework(TDF). Interviews were transcribed and refined the theme using directed content and induction analysis.Using the TDF as the initial coding framework, the themes were then merged into the most relevant domains.Finally, the correspondence between theoretical domains and the healthy behavior of stroke patients was determined based on the frequency and relationship between themes.Results:This study identified nine theoretical domains that affected the healthy behavior of stroke patients: knowledge, skills, motivation and goals, social influences, social/professional role and identity, environment context and resources, belief about capability, consequence belief and behavioral regulation.Conclusion:The healthy behavior of stroke patients is complex and influenced by several factors.The nine theoretical domains identified in this study will provide recommendations for future healthy behavior interventions for stroke patients.

Objective:To investigate the predictive value of thrombus permeability for poor outcome and symptomatic intracranial hemorrhage (sICH) after endovascular therapy (EVT) in patients with middle cerebral artery occlusion stroke.Methods:Patients with middle cerebral artery occlusion stroke underwent EVT at the Cardiocerebrovascular Disease Hospital, the Affiliated Hospital of Yan'an University from January 2018 to January 2024 were included retrospectively. Thrombus attenuation increase (TAI) was used to evaluate thrombus permeability. The poor outcome was defined as a modified Rankin Scale score >2 at 90 days after procedure. sICH was defined as an increase of ≥4 of the National Institutes of Health Stroke Scale (NIHSS) score relative to baseline or lowest within 7 days after EVT, and CT scan showed cerebral hemorrhage. Multivariate logistic regression analysis was used to determine the independent influencing factors of poor outcome and sICH. Receiver operating characteristic (ROC) curves was used to evaluate the predictive value of TAI for poor outcome and sICH. Results:A total of 77 patients with middle cerebral artery occlusion stroke received EVT treatment were enrolled, including 44 males (57.1%), aged 62.1±12.4 years. Thirty-three patients (48.1%) had poor outcome, 35 (45.5%) experienced hemorrhagic transformation, of which 12 (15.6%) were sICH. Multivariate logistic regression analysis showed that TAI (odds ratio [ OR] 0.930, 95% confidence interval [ CI] 0.883-0.980; P=0.007) and sICH ( OR 0.868, 95% CI 0.784-0.961; P=0.006) were the independent influencing factors of poor outcome. ROC curve analysis showed that the area under the curve of TAI for predicting poor outcome at 90 days was 0.836 (95% CI 0.742-0.930). The cut-off value was 10.135 HU. The sensitivity and specificity were 73.0% and 92.5%, respectively. The area under the curve of TAI for predicting sICH was 0.750 (95% CI 0.637-0.902). The cut-off value was 18.200 HU. The sensitivity and specificity were 94.1% and 64.5%, respectively. Conclusions:TAI has certain predictive value for poor outcome and sICH after EVT in patients with middle cerebral artery occlusion stroke. Patients with higher thrombus permeability are less likely to develop sICH after EVT and have a higher likelihood of good outcome.

OBJECTIVETo compare the efficacy and adverse events of adjusted BACOD (bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone) regimen (continuous intravenous infusion) and conventional BACOD regimen (conventional intravenous drip) in the treatment of relapsed and refractory diffuse large B cell lymphoma (DLBCL).

METHODSRetrospective analysis of 63 cases of relapsed or refractory DLBCL patients was performed, 32 patients received conventional BACOD regimen and 31 patients received adjusted BACOD regimen.

RESULTSThe response rates for adjusted group and conventional group were 87.1%(27/31)and 62.5%(20/32), respectively, during a median follow-up of 14(7-84) months. The difference was statistically significant between the two groups (P=0.025). The main adverse events were myelosuppression, gastrointestinal adverse reactions were rarely serious, and there were no serious liver and kidney toxicity. The median overall survival (OS) was 33 months for adjusted group and 12 months for conventional group, there was statistical differences (P=0.019). The median progression free survival (PFS) was 11 months and 8 months for two groups, the difference was not statistically significant (P=0.095). 1-year survival rates were 68.8% for adjusted group and 44.3% for conventional group, there were no statistical differences (P=0.055). The expected 3- and 5-year survival rates of adjusted group were significantly higher than that of conventional group (47.1% vs 12.8%, P=0.002; 37.7% vs 8.5%, P=0.006, respectively).

CONCLUSIONCompared with the conventional BACOD regimen, the adjusted BCOAD regimen is effective and well tolerated in patients with relapsed or refractory DLBCL, the overall response rate and OS increased.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; Bleomycin ; Cyclophosphamide ; Dexamethasone ; Doxorubicin ; Female ; Humans ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Vincristine