Despite the remarkable clinical efficacy achieved by immune checkpoint blockade(ICB)and adoptive T cell therapy,the majority of patients with solid tumors still fail to achieve long-term responses to immunotherapy.One important reason is the complex metabolic patterns and inhibitory signals within the tumor microenvironment(TME),which induce metabolic reprogramming in immune cells and thereby impair their anti-tumor efficacy.This review summarizes the metabolic preferences of CD8+T cells across various differentiation states,explores the metabolic changes that occur during their interaction with tumor cells and the TME,and discusses how these metabolic changes affect differentiation,function,and stemness of CD8+T cells.Additionally,strategies that target metabolic molecules or pathways are highlighted to enhance the antitumor ability of CD8+T cells,thereby improving the efficacy of chimeric antigen receptor gene-modified T lymphocyte(CAR-T cell)immunotherapy and ICB therapy.

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 investigate the effect of bone morphogenetic protein-9(BMP9)overexpression on inflammatory response and apoptosis of alveolar epithelial cells induced by lipopolysaccharide(LPS).Methods:A549 cells were stimulated with 0.1 μg/ml LPS,expressions and changes of BMP9 protein at different time points(LPS stimulation for 0 h,6 h,12 h,24 h)were detected by Western blot.Expression of BMP9 in A549 cells was up-regulated by transfection of BMP9 plasmid,and the transfection efficiency was verified by Western blot and qPCR.After 12 h of LPS stimulation,levels of inflammatory factors TNF-α,IL-6,IL-1β in cell supernatant were detected by ELISA,expressions of anti-apoptotic protein(Bcl-2)and pro-apoptotic protein(Bax)were detected by Western blot,and apoptosis of cells was detected by TUNEL staining.Results:With the extension of LPS stimulation time,expression of BMP9 was down-regulated.Overexpression of BMP9 successfully up-regulated expression of BMP9 in A549 cells.LPS stimulation promoted secretions of TNF-α,IL-6 and IL-1β from A549 cells,increased apoptosis,promoted Bcl-2 expression while inhibited Bax expression.Overexpression of BMP9 inhibited TNF-α,IL-6 and IL-1β releasing,decreased apoptosis,inhibited Bcl-2 expression,while promoted Bax expression.Conclusion:In LPS-stimulated A549 cells,BMP9 expression is gradually decreased at a time-depen-dent manner.Overexpression of BMP9 can inhibit LPS-induced inflammatory response and apoptosis in A549 cells.

BACKGROUND:Some research has suggested that regulation of gut microbiota may influence the course of intervertebral disc degeneration.However,the causal relationship of gut microbiota on intervertebral disc degeneration is unknown.OBJECTIVE:To assess the potential causal relationship between gut microbiota and intervertebral disc degeneration using a Mendelian randomization method.METHODS:Genome-wide association analysis summary statistics for 473 gut microbiota and genome-wide association analysis summary data for intervertebral disc degeneration from the R11 of the FinnGen database(46 205 cases of intervertebral disc degeneration and 322 314 controls)from the most recent publicly available publication were applied.Inverse variance weighting,MR-Egger regression,weighted median,weighted modeling,and simple modeling were used to investigate the causal relationship between gut microbiota and intervertebral disc degeneration.Sensitivity analyses were used to test whether the results of Mendelian randomization analyses were reliable.Reverse Mendelian randomization was performed with all gut microbiota as the outcomes for effect analysis and sensitivity analysis.RESULTS AND CONCLUSION:(1)The results of the inverse variance weighting method of the forward Mendelian randomization method showed that the order Trichosporonaceae,the family UBA-6960,the family Anaerobes thermophilus,the family Salmonellaceae,the genus Pseudomonas tufts,the species Gordonella and the species Euclidia showed a positive correlation with intervertebral disc degeneration.The order Spirochaetes,the order Pseudomonas,the family Spirochaetaceae,the genus CAG-776,the genus Helicobacter,the species CAG-448 sp003150135,the species CAG-776 sp000438195,the species Brautella-A sp000285855 and the species Hanson's Brautella showed a negative correlation with intervertebral disc degeneration.(2)The results of reverse Mendelian randomization showed that intervertebral disc degeneration was positively correlated with the genus Bartonella rosea,the genus Geobacillus C,the species Escherichia fumigatus,the species Propionibacterium fumigatus,the species UBA-1777 sp900319835,the species Pseudomonas aeruginosa and the species Bacillus subtilis,while negatively correlated with the species Streptomyces mingoldii,the species Prevotella sp000434975,the species Brault's A sp000285855,the species CAG-194 sp002441865 and the species CAG-590 sp000431135.(3)No heterogeneity or horizontal pleiotropy was found in the two-way sensitivity analysis.(4)The results described above indicate that the causal relationship between gut microbiota and intervertebral disc degeneration based on the Finnish database contributes to the exploration on new biomarkers for the early prediction and treatment of intervertebral disc degeneration in clinical practice.In addition,the establishment of a large database and the integration of medical data from multiple centers can be drawn upon in biomedical research in China to provide a solid foundation for studying the relationship between gut microbiota and intervertebral disc degeneration.We will strengthen communication and cooperation with research teams in other countries to jointly promote the research on the relationship between gut microbiota and diseases and contribute to the development of global medicine.

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.

BACKGROUND:Some research has suggested that regulation of gut microbiota may influence the course of intervertebral disc degeneration.However,the causal relationship of gut microbiota on intervertebral disc degeneration is unknown.OBJECTIVE:To assess the potential causal relationship between gut microbiota and intervertebral disc degeneration using a Mendelian randomization method.METHODS:Genome-wide association analysis summary statistics for 473 gut microbiota and genome-wide association analysis summary data for intervertebral disc degeneration from the R11 of the FinnGen database(46 205 cases of intervertebral disc degeneration and 322 314 controls)from the most recent publicly available publication were applied.Inverse variance weighting,MR-Egger regression,weighted median,weighted modeling,and simple modeling were used to investigate the causal relationship between gut microbiota and intervertebral disc degeneration.Sensitivity analyses were used to test whether the results of Mendelian randomization analyses were reliable.Reverse Mendelian randomization was performed with all gut microbiota as the outcomes for effect analysis and sensitivity analysis.RESULTS AND CONCLUSION:(1)The results of the inverse variance weighting method of the forward Mendelian randomization method showed that the order Trichosporonaceae,the family UBA-6960,the family Anaerobes thermophilus,the family Salmonellaceae,the genus Pseudomonas tufts,the species Gordonella and the species Euclidia showed a positive correlation with intervertebral disc degeneration.The order Spirochaetes,the order Pseudomonas,the family Spirochaetaceae,the genus CAG-776,the genus Helicobacter,the species CAG-448 sp003150135,the species CAG-776 sp000438195,the species Brautella-A sp000285855 and the species Hanson's Brautella showed a negative correlation with intervertebral disc degeneration.(2)The results of reverse Mendelian randomization showed that intervertebral disc degeneration was positively correlated with the genus Bartonella rosea,the genus Geobacillus C,the species Escherichia fumigatus,the species Propionibacterium fumigatus,the species UBA-1777 sp900319835,the species Pseudomonas aeruginosa and the species Bacillus subtilis,while negatively correlated with the species Streptomyces mingoldii,the species Prevotella sp000434975,the species Brault's A sp000285855,the species CAG-194 sp002441865 and the species CAG-590 sp000431135.(3)No heterogeneity or horizontal pleiotropy was found in the two-way sensitivity analysis.(4)The results described above indicate that the causal relationship between gut microbiota and intervertebral disc degeneration based on the Finnish database contributes to the exploration on new biomarkers for the early prediction and treatment of intervertebral disc degeneration in clinical practice.In addition,the establishment of a large database and the integration of medical data from multiple centers can be drawn upon in biomedical research in China to provide a solid foundation for studying the relationship between gut microbiota and intervertebral disc degeneration.We will strengthen communication and cooperation with research teams in other countries to jointly promote the research on the relationship between gut microbiota and diseases and contribute to the development of global medicine.

Objective:To investigate the impact of different treatment sequences of immunotherapy combined with chemoradiotherapy (CRT) as the first-line therapy on the prognosis of patients with stage III non-small cell lung cancer (NSCLC).Methods:Clinical data of 112 patients with stage III NSCLC treated at the Fourth Hospital of Hebei Medical University from January 2019 to December 2021 were retrospectively collected, with follow-up continued until December 31, 2023. According to the sequence of CRT and immune checkpoint inhibitors (ICIs) therapy, patients were divided into 3 groups: ICIs simultaneous with CRT (sICR, n=20), chemotherapy combined with ICIs followed by CRT (CI-CR, n=53), and CRT followed by consolidative ICIs (CR-I, n=39). Analyses were performed before and after propensity score matching (PSM). Survival outcomes were assessed using the Kaplan-Meier method and compared by log-rank tests, and prognostic factors were identified through multivariate Cox regression analysis. Results:The median overall survival (OS) and progression-free survival (PFS) for the entire cohort were 30.1 months (95% CI: 21.4-38.9) and 12.8 months (95% CI: 9.14-16.1), respectively. Before PSM: No significant differences were observed in OS and PFS among the 3 groups ( χ2=0.18, 1.05; P=0.669, 0.305). However, OS in the sICR and CR-I groups was significantly better than that in the CI-CR group ( χ2=4.43, 6.11; P=0.035, 0.013). After PSM: Each group included 17 patients. There were no significant differences in OS or PFS among the 3 groups ( χ2=2.50, 2.74; P=0.287, 0.254), and pairwise comparisons also showed no significant differences. Multivariate Cox regression analysis revealed that clinical stage ( HR=3.392, 95% CI: 1.215-9.470, P=0.020), number of immunotherapy cycles ( HR=0.312, 95% CI: 0.100-0.972, P=0.044), and treatment response ( HR=6.566, 95% CI: 1.705-25.284, P=0.006) were independent prognostic factors for OS. After PSM, the numbers of patients with grade ≥2 treatment-related adverse events were 13 in the sICR group, 10 in the CI-CR group, and 9 in the CR-I group, with no significant differences among them ( χ2=2.181, P=0.336). Conclusions:First-line immunotherapy combined with chemoradiotherapy showed favorable clinical efficacy in locally advanced NSCLC compared to other studies, but the treatment sequence did not significantly affect prognosis. It is recommended that immunotherapy be administered for at least four cycles.

In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.

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