Hemoglobin A1c (HbA1c) has a unique structure that makes monoclonal antibody (mAb) preparation challenging. This study aims to develop a method for preparing HbA1c mAbs and establish a fluorescent immunochromatographic assay (FICA) for rapid detection of HbA1c. Three glycosylated peptides were synthesized and used to prepare complete antigens, which were identified by dot enzyme-linked immunosorbent assay (Dot-ELISA) and ultraviolet absorption spectroscopy. The complete antigens and natural HbA1c were used for cross-immunization of mice, and the optimal complete antigen was selected. The mouse with the highest serum titer was chosen for mAb preparation. The purity and specificity of the mAbs were verified, and a FICA method was developed. The optimal complete antigen, with a titer of 1:512 000, was successfully prepared and selected. Fusion with splenocytes resulted in four specific HbA1c antibodies (purity > 90%). The best antibody exhibited a binding constant (Ka) of 1.67×10¹⁰ L/mol with the antigen. Based on this antibody, a FICA method was successfully established, capable of producing results within 15 min. The method demonstrated a good linear range (3%-13% HbA1c, y=0.071 3x+0.005 6, R²=0.993 7), recovery rates of 98%-102%, precision < 10.00%, and no nonspecific reactions. Clinical testing of 210 samples showed positive agreement of 96.36%, negative agreement of 97.00%, and overall agreement of 96.68%. The receiver operating characteristic (ROC) curve analysis yielded an area under curve (AUC) of 0.980 9 [95% confidence interval (CI): 0.961 0-1.000 0], with high consistency verified in multicenter studies. We successfully developed a key technique for preparing HbA1c monoclonal antibodies and established a FICA method for rapid detection of HbA1c. It will provide an efficient and convenient detection method for the early diagnosis and long-term management of diabetes and its complications.
Antibodies, Monoclonal/biosynthesis* ; Animals ; Mice ; Glycated Hemoglobin/immunology* ; Mice, Inbred BALB C ; Humans ; Antibody Specificity ; Chromatography, Affinity/methods* ; Enzyme-Linked Immunosorbent Assay/methods* ; Female

Background and aims:Early and accurate diagnosis of the coexistence of Wilson's disease(WD)and chronic hepatitis B(CHB)presents a significant challenge for clinicians.The objective of this study was to retrospectively analyse the characteristics of such patients to improve clinical practice and provide a reference for clinical management.Methods:From January 2011 to December 2022,35 patients with concurrent CHB and WD(CHB+WD group)were identified.A total of 127 patients with CHB(CHB group)and 168 patients with WD(WD group)were included in the control group between January 2016 and December 2021.Propensity score matching(PSM)was performed to balance the baseline values between groups.The Kaplan-Meier(K-M)survival analysis and log-rank test were performed to compare the prognoses.Results:In the cohort of 35 patients with concurrent CHB and WD,74.3％of patients(26 patients)faced a substantial delay of up to 10 years(range:0-40 years)in WD diagnosis following their CHB diagnosis.Twenty-three(65.7％)patients had cirrhosis at the time of WD diagnosis,and 26(74.3％)patients experienced liver failure.The levels of serum copper and uric acid were lower in patients in the CHB+WD group than in those in the CHB group.Patients in the CHB+WD group presented higher alanine transaminase and total bile acid levels compared to those in the WD group.K-M survival analysis indicated that patients with CHB and WD had poorer outcomes than those with CHB alone;however,the outcomes were similar to those of individuals with WD alone.The optimal cut-point of serum ceruloplasmin(CP)in identifying WD in CHB patients was 0.10 g/L before PSM and after PSM.Conclusions:The present study emphasizes the importance of clinicians being vigilant for concurrent CHB and WD diagnoses,as delays in WD diagnosis may adversely affect patient outcomes.CHB patients with serum CP below 0.10 g/L are highly recommended to screen for WD.

Objective To investigate the correlation between the epidermal growth factor receptor(EGFR)gene polymorphisms and non-small cell lung cancer(NSCLC)in Yunnan Han population.Methods A total of 407 NSCLC patients and 526 healthy controls were included.Five SNPs(rs1050171,rs2072454,rs2227983,rs1140475 and rs2293347)in EGFR were genotyped using TaqMan assay.The association between SNPs and NSCLC,as well as pathological types,were analyzed.Results In dominant mode,rs2072454 C/T-T/T may be a risk factor for NSCLC(P=0.004;OR=1.50,95%CI 1.14～1.96).There was a significant difference in genotype frequency between the SCC group and the control group(P=0.007),but no difference in allele frequency after Bonferroni correction(P>0.01);In the dominant mode,C/T-T/T at this locus relative to C/C was a risk for AC(P=0.002;OR=1.86,95%CI 1.24～2.80).rs1050171 A/A had a significantly higher risk of SCC in recessive mode(P=0.006,OR=2.66,95%CI 1.33～5.33).In dominant mode,rs2227983 A/G-G/G/G was a risk factor for the development of SCC relative to A/A(P=0.007;OR=1.83,95%CI 1.15～2.89).Conclusion The SNPs rs2072454,rs1050171 and rs2227983 in EGFR gene may be associated with the risk of NSCLC development as well as the type of pathology in Yunnan Han population.

Objective To investigate the association between four single nucleotide polymorphisms(SNP)(rs9340 in MAPK1,rs14804 in NRAS,rs712 and rs7973450 in KRAS)in the 3'UTR of ERK1/2 signaling pathway-related genes and non-small cell lung cancer(NSCLC).Methods A total of 478 NSCLC patients and 480 healthy controls were enrolled in this study.Four SNPs were genotyped by using TaqMan assays.The association between the four SNPs and NSCLC was analyzed.Results The distribution frequency difference of the allele of rs9340 was statistically significant between the control group and the non-small cell squamous cell carcinoma(SCC)group(P = 0.009),suggesting that the G allele of rs9340 may be a protective factor for non-small cell lung squamous cell carcinoma(OR = 0.67,95%CI:0.50～0.91).In addition,in the<50 years age group,the distribution frequency difference of the allele of rs9340 was statistically significant between the control group and the NSCLC group(P = 5.07×10-4),indicating that the G allele of rs9340 may be a protective factor for NSCLC(OR = 0.46,95%CI:0.29～0.72).Conclusion The SNP rs9340 in MAPK1 may be associated with the risk of NSCLC.

Objective To investigate how the immune status,inflammatory factors,and nervous function of coma patients with severe traumatic brain injury(sTBI)were impacted by early personalized enteral nutrition support in combination with An'gong Niuhuang pill.Methods A total of 80 patients with sTBI admitted to the First Central Hospital of Baoding from July 2020 to June 2023 were selected as the study objects.The patients were divided into control group and observation group according to different treatment methods,with 40 cases in each group.The control group received early individualized nutritional support treatment,and the observation group was supplemented with one An'gong Niuhuang pill daily(3 g per pill)based on the above nutritional support.Each group underwent a 7-day treatment regimen.Serum immunoglobulin G(IgG),immunoglobulin A(IgA),immunoglobulin M(IgM)and lymphocyte subsets were collected at 1,3 and 7 days after treatment,as well as serum levels of interleukin-6(IL-6),neutrophil count(NEUT)and lymphocyte count(LYM)before and 7 days after treatment,neutrophil/lmphocyte ratio(NLR)was calculated,and the change of nerve function was observed.Results ①Complement and immunoglobulin:the level of complement C4 in the observation group was significantly lower than that in the control group 3 days after treatment(g/L:0.2±0.1 vs.0.3±0.1,P<0.05),and the level of complement C3,IgA,IgG and IgM were significantly higher than that in the control group 7 days after treatment[C3(g/L):1.2±0.2 vs.0.9±0.2,IgA(g/L):2.7±0.8 vs.2.0±0.6,IgG(g/L):9.3±1.2 vs.8.0±1.0,IgM(g/L):1.2±0.4 vs.0.7±0.3,all P<0.05].②Lymphocyte subsets:the levels of CD8+T and natural killer cell(NK cell)in the observation group were significantly lower than those in the control group at 3 days after treatment(CD8+T:0.20±0.05 vs.0.25±0.06,NK cell:0.16±0.10 vs.0.22±0.03,both P<0.05),the levels of CD3+T and CD4+T in the observation group were significantly higher than those in the control group 7 days after treatment(CD3+T:0.76±0.07 vs.0.71±0.01,CD4+T:0.48±0.05 vs.0.41±0.07,both P<0.05),NK cell levels continued to decrease compared with control group(0.12±0.05 vs.0.17±0.02,P<0.05).③Inflammation index and blood routine index:after 7 days of treatment,IL-6 and NLR levels in both groups were significantly decreased compared with those before treatment,and the decrease was more significant in the observation group[IL-6(ng/L):26.4(15.3,38.4)vs.42.9(30.7,58.8),NLR:5.7±2.5 vs.9.1±5.0,both P<0.01],the level of LYM in both groups after treatment was significantly lower than that before treatment,but the level of LYM in the observation group was significantly higher than that in the control group after treatment(×109/L:1.6±0.4 vs.1.1±0.5,P<0.05).④GCS score:7 days after treatment,the GCS score of both groups was significantly higher than before treatment,but the GCS score of the observation group was significantly higher than that of the control group(6.8±2.7 vs.5.6±2.1,P<0.05).Conclusion Treatment with An'gong Niuhuang pill based on early individualized enteral nutrition support can effectively reduce the inflammatory response of patients with sTBI,enhance the body fluid and cellular immune function,and help to improve the neurological function.

Objective To investigate the effect and mechanism of musk-containing serum on the migration of bone marrow mesenchymal stem cells(BMSCs).Methods Sixty SD rats were randomly divided into four groups:musk-high-,medium-and low-dose groups and blank control group;medicated serum was prepared.Fifteen SD rats were isolated and cultured with BMSCs,and the third generation of BMSCs were identified by morphology,phenotype,osteogenic and adipogenic induction.BMSCs received medicinal healing intervention with high-,medium-and low-(16.8,8.4,and 4.2 μL/100 g)musk,and the cell proliferation rate was detected by MTT assay.Under the intervention of the protein kinase C(PKC)signaling pathway(GF109203X),the effect of musk with pharmacition on the migration of BMSCSs was detected with the Transwell test.Results The rat BMSCs were attached to the wall,with orderly arrangement and good cell viability.Phenotypic identification revealed that the expressions of CD44 and CD90 were positive,while the expressions of CD45 and CD34 were negative,and the cells could differentiate into osteoblasts and adipocytes.The proliferation rates of BMSCSs with different concentrations at different time periods were higher than those in the blank control group(P＜0.05).The number of BMSCs in the low-concentration musk group(4.2 μL/100 g)was significantly increased at 24 h,48 h and 72 h after the addition of the blocking agent GF109203X(P＜0.05).The migration quantity of the low-concentration musk group+blocker group(GF109203X)significantly decreased at different time periods,and there was no significant difference between different time groups(P＞0.05).Conclusion The mechanism of musk-containing serum in promoting BMSCs migration may be related to the activation of PKC signaling pathway.

This study aims to establish a time-resolved fluorescence immunochromatography method for simultaneous determination of human papillomavirus (HPV) type 16 E6 and L1 protein concentrations. The amount of lanthanide microsphere-labeled antibodies, the concentration of coated antibodies, and the reaction time were optimized, and then a test strip for the simultaneous determination of the protein concentrations was prepared. The performance of the detection method was evaluated based on the concordance of the results from clinical practice. The optimal conditions were 8 μg and 10 μg of HPV16 L1 and E6-labeled antibodies, respectively, 1.5 mg/mL coated antibodies, and reaction for 10 min. The detection with the established method for L1 and E6 proteins showed the linear ranges of 5-320 ng/mL and 2-64 ng/mL and the lowest limits of detection of 1.78 ng/mL and 1.09 ng/mL, respectively. There was no cross reaction with human immunodeficiency virus (HIV), treponema pallidum (TP), or HPV18 E6 and L1 proteins. The average recovery rate of the established method was between 97% and 107%. The test strip prepared in this study showed the sensitivity, specificity, and diagnostic accuracy of 97.46%, 90.57%, and 95.32%, respectively, in distinguishing patients with cervical cancer and precancerous lesions from healthy subjects, with the area under the curve (AUC) of 0.980 1 and 95% Confidence Interval (CI) of 0.956 5 to 1.000 0. The time-resolved fluorescence immunochromatography combined with the test strips prepared in this study showed high sensitivity, high accuracy, simple operation, and rapid reaction in the quantitation of HPV16 E6 and L1 proteins. It thus can be used as an auxiliary method for the diagnosis and early screening of cervical cancer and precancerous lesions and the assessment of disease course.
Oncogene Proteins, Viral/immunology* ; Humans ; Chromatography, Affinity/methods* ; Female ; Human papillomavirus 16 ; Repressor Proteins/immunology* ; Capsid Proteins/immunology* ; Papillomavirus Infections/diagnosis* ; Fluorescence ; Uterine Cervical Neoplasms/virology*

Objective To develop engineered bacterial membrane biomimetic nanoparticles,Angiopep-2 E.coli membrane(ANG-2 EM)@PDA-PEI-CpG(ANG-2 EM@PPC),for efficient targeted drug delivery in the treatment of glioma,and to provide theoretical and technical support for targeted glioma therapy.Methods The expression of inaX-N-angiopep-2 engineered bacteria was constructed in the laboratory,and ANG-2 EM was obtained through lysozyme treatment and ultrafiltration centrifugation.ANG-2 EM@PPC was prepared by ultrasonication of bacterial membranes.Western blotting,agarose gel electrophoresis,and transmission electron microscopy(TEM)were used to verify the preparation.Particle size and Zeta potential were measured to investigate the stability of ANG-2 EM@PPC.Regarding cell experiments,CCK-8 assay was performed to determine the effect of ANG-2 EM@PPC on the survival rate of neutrophils.A flow chamber model was designed and constructed,and the uptake efficiency of neutrophils was measured by flow cytometry to investigate the hitchhiking efficiency of ANG 2 EM@PPC on neutrophils in inflammatory environment.Neutrophil death patterns were characterized by fluorescence microscopy,and flow cytometry and Western blotting were performed to examine neutrophil apoptotic bodies and the proportion of apoptotic bodies produced.Regarding animal experiments,a mouse model of in situ glioma was established and the inflammatory environment of tumor tissue was verified.The tumor model mice were divided into three groups,including DiR group,EM@PPC group,and ANG-2 EM@PPC group(all n=3),which were injected with DiR,ANG-2 EM@PDA-PEI-CpG,and EM@PDA-PEI-CpG via the tail vein,respectively(all at 10 mg/kg).Fluorescence images of organs and the brain were used to examine the distribution of the three formulations in vivo and in the brain.The tumor model mice were further divided into PBS group,PDA group,PC group,PPC group,EM@PPC group,and ANG-2 EM@PPC group(all n=4),which were injected with PBS,PDA,PC,PPC,EM@PPC,and ANG-2 EM@PPC injected via the tail vein,respectively(all at 10 mg/kg).Imaging was performed in vivo to observe tumor regression,and the survival rate and body mass of mice were measured to evaluate in vivo pharmacodynamics.TUNEL staining(brain tissue)and HE staining(brain,heart,liver,spleen,lung and kidney tissues)were performed to evaluate the therapeutic effect.Results The results of TEM showed successful preparation of engineered bacterial membrane biomimetic nanoparticles,with PPC exhibiting a distinct shell-core structure and a shell thickness of about 8.2 nm.Due to the coating of ANG-2 EM,the shell thickness of ANG-2 EM@PPC increased to about 9.6 nm,with a clear bacterial membrane layer on the surface.Stability was maintained for at least one week.ANG-2 EM@PPC had no significant effect on the activity of neutrophils according to the findings from the CCK-8 assay.Flow cytometry showed that ANG-2 EM@PPC uptake is enhanced in activated neutrophils and hitchhiking on neutrophils was more efficient in the stationary state than that in the flowing condition.Compared with the EM@PPC group,the neutrophil hitchhiking ability of the ANG-2 EM@PPC group was enhanced(uptake efficiency 24.9%vs.31.1%).Fluorescence microscopy showed that ANG-2 EM@PPC changed the death pathway of neutrophils from neutrophil extracellular traps-osis(NETosis)to apoptosis.Western blot confirmed the production of neutrophil apoptotic bodies,and flow cytometry showed that the production rate was as high as 77.7%.Animal experiments showed that there was no significant difference in the distribution of engineered bacterial membrane biomimetic nanoparticles in the organs(heart,liver,spleen,lungs,and kidney)in the DiR group,the EM@PPC gropu,and the ANG-2 EM@PPC group(P＞0.05),but there was higher distribution in the brain tissue in EM@PPC and ANG-2 EM@PPC groups compared to the DiR group(P＜0.05).Engineered bacterial membrane biomimetic nanoparticles crossed the blood-brain barrier(BBB),and exhibited high affinity to and internalization by neutrophils located in brain tumors.Compared with PBS,PDA,PC,and PPC groups,the survival rate and body mass of mice in the EM@PPC group were improved,tumor fluorescence intensity was weakened,and apoptotic cells were increased.These trends were even more prominent in the ANG-2 EM@PPC group.No abnormality was found in the HE staining of any group.Conclusion An ANG-2 EM@PPC nanodelivery system with inflammation response characteristics was successfully prepared,capable of crossing BBB and targeting the tumor inflammatory microenvironment to improve the anti-glioma efficacy.This study provides a new drug delivery strategy for glioma treatment and offers a new idea for targeted drug delivery in the non-invasive inflammatory microenvironments in other central nervous system diseases.

Objective:To explore the effects of direct antiviral agent (DAAs) on the frequency of peripheral blood mononuclear cells and their activating factors sCD14s and CD163 in patients with chronic hepatitis C.Methods:Data of 15 treatment-naive chronic hepatitis C patients and 10 healthy controls were collected. Patients with chronic hepatitis C were treated with DAAs for 12 weeks. Blood samples were collected at 0, 4 and 12 weeks respectively, and blood samples of healthy controls were used as controls. Flow cytometry was used to detect the frequency of classical CD14 ++CD16 - mononuclear cells and pro-inflammatory CD14 +CD16 + mononuclear cells in peripheral blood. Serum sCD14s and sCD163 were detected by enzyme-linked immunosorbent assay. The comparison between the two groups was performed by t-test. The comparison between multiple groups was performed by analysis of variance, and further pairwise comparison was performed by LSD-t test. Results:Prior DAAs treatment, peripheral blood CD14 +CD16 + mononuclear cell frequency (18.49% ± 1.54% vs. 10.65% ± 0.83%), serum sCD14s [(64 407.38 ± 5778.49) pg/ml vs. (28 370.76 ± 2 357.68 ) pg/ml] and sCD163 [(22 853.80 ± 4 137.61) pg/ml vs. (2 934.41 ± 223.31) pg/ml] were all higher than healthy controls ( P < 0.05), while the frequency of CD14 ++CD16 - mononuclear cells in peripheral blood was lower than healthy controls (59.14%±0.54% vs. 72.75%±1.31%, P < 0.01). During DAAs treatment, CD14 +CD16 + mononuclear cells frequency, serum sCD14 and sCD163 were all decreased significantly. After 12 weeks of treatment, CD14 +CD16 + mononuclear cells had decreased to nearly normal level (12.42% ± 1.60% vs. 10.65% ± 0.83%, P > 0.05), and serum sCD14 and scd163 were still higher than those of healthy controls [sCD14: (44 390.06 ± 3 330.17) pg / ml vs. (28 370.76 ± 2 357.68) pg/ml, Scd163: (11 494.79 ± 1 836.97) pg / ml vs. (2 934.41 ± 223.31) pg / ml, P < 0.01], while the frequency of CD14 ++CD16 -mononuclear cells had gradually increased during the course of treatment and neared healthy control level after 12 weeks of treatment. There was no statistically significant difference between the two groups (71.54) % ± 2.99% vs. 72.75% ± 1.31%, P > 0.05). Conclusion:DAAs therapy can reduce the activation of peripheral blood mononuclear cells in patients with chronic hepatitis C.

Objective: To investigate the effects of direct-acting antiviral agents (DAA) therapy on the frequency of myeloid-derived suppressor cells (MDSC) and their subset of monocytic myeloid-derived suppressor cells (M-MDSC) in chronic hepatitis C (CHC) patients. Methods: A total of 32 treatment-naive CHC patients and 16 healthy controls were recruited at Third Affiliated Hospital of Sun Yat-Sen University from June 2016 to June 2017. The peripheral blood mononuclear cells (PBMC) were separated from the peripheral blood of patients with CHC before DAA therapy, at four weeks after DAA therapy, at 12 weeks after DAA therapy and 12 weeks after the end of DAA therapy. The frequencies of MDSC and M-MDSC were detected by the flow cytometer. The t test, U test and chi-square test was employed to analyze the data. Results: All the 32 treatment-naive patients achieved the rapid virological response and no virological breakthrough was observed. Before DAA therapy, the frequency of MDSC in CHC patients was 2.18%, which was higher than healthy individuals (0.60%; Z=-4.593, P<0.01), and positively correlated with the plasma levels of hepatitis C virus (HCV) RNA (r=0.688, P<0.01) and aspartate aminotransferase (r=0.735, P<0.01). After four weeks of DAA therapy, the frequency of MDSC decreased significantly to 1.07%, with no statistical significance compared to the controls (Z=-1.221, P>0.05). However, at 12 weeks after DAA therapy, the MDSC frequency increased, with statically significance compared to the controls (1.64% vs 0.60%, Z=-3.117, P=0.002). At 12 weeks after the end of DAA therapy, the MDSC frequency had decreased to 1.29% again, with no statistical significance compared to the controls (Z=-1.387, P=0.664). The changes of M-MDSC frequency were slightly different. Before DAA therapy, the frequency of M-MDSC in CHC patients was higher compared to healthy controls (1.66% vs 0.81%, Z=-2.745, P<0.01). The frequencies of M-MDSC were 0.91%, 1.09% and 1.10% at four, 12 weeks after DAA and 12 weeks after the end of DAA therapy, respectively. The differences were not statistically significant compared to the controls (Z=-0.589, -1.028 and -0.486, respectively, all P>0.05). Conclusion Immune status of the peripheral MDSC and M-MDSC can return to normal after DAA therapy in CHC patients.