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

Objective To explore the relationship between triglyceride-glucose index(TyG)and acute ischemic stroke with large vessel occlusion(AIS-LVO)of anterior circulation.Methods A retrospective study was conducted on patients with anterior circulation AIS-LVO who underwent emergency endovascular thrombectomy at Neurovascular Center of The First Affiliated Hospital of Naval Medical University from Jan.2018 to Dec.2019.According to modified Rankin scale(mRS)score 90 d after operation,the patients were assigned to favorable outcome group(mRS score 0-2)or unfavorable outcome group(mRS score 3-6),and the TyG was compared.According to the median of TyG,the patients were assigned to low-TyG group(TyG＜8.57)or high-TyG group(TyG ≥8.57),and the clinical data,laboratory indexes,and imaging characteristics were compared.Receiver operating characteristic curve was used to evaluate the predictive value of TyG for poor prognosis.Results A total of 135 patients were enrolled,with 72 in the favorable outcome group and 63 in the unfavorable outcome group.The TyG of the unfavorable outcome group was significantly higher than that of the favorable outcome group(8.82+0.63 vs 8.43+0.60,P＜0.001).There were 67 patients in the low-TyG group and 68 in the high-TyG group.Compared with the low-TyG group,the proportion of patients with hyperlipidemia history(P=0.003),systolic blood pressure at admission(P=0.018),fasting blood glucose level(P＜0.001),and triglyceride level(P＜0.001)were significantly higher in the high-TyG group,the infarct core volume was significantly larger(P=0.025),the high density lipoprotein-cholesterol level was significantly lower(P=0.013),and the mRS score 90 d after operation was significantly higher(3[1,5]vs 1[0,5],P=0.049).The TyG had certain predictive value for poor prognosis in anterior circulation AIS-LVO patients(area under curve value=0.662,95％confidence interval 0.571-0.753).Conclusion TyG is elevated in anterior circulation AIS-LVO patients with poor prognosis,and may be a potential prognostic indicator for anterior circulation AIS-LVO patients.

Objective To investigate the safety and effectiveness of endovascular treatment in patients with acute ischemic stroke due to large vessel occlusion(AIS-LVO)over 24 h after onset.Methods The clinical data of AIS-LVO patients who received endovascular treatment in Neurovascular Center of The First Affiliated Hospital of Naval Medical University from Jan.2018 to Dec.2022 were retrospectively analyzed,including baseline characteristics,imaging findings,treatment,degree of vascular recanalization(modified thrombolysis in cerebral infarction grade 2b and 3 for successful recanalization)and prognosis.Results A total of 57 patients were included,including 42 males and 15 females,aged from 30 to 84 years old.The most common risk factors were hypertension(39 cases,68.4%),followed by smoking history(24 cases,42.1%),diabetes mellitus(17 cases,29.8%),previous stroke history(16 cases,28.1%),and atrial fibrillation(9 cases,15.8%).Before treatment,the National Institutes of Health stroke scale score was 12.84±7.04,and the Alberta Stroke Program early computed tomography score was 9.00(7.00,10.00).Vascular occlusion sites included middle cerebral artery occlusion in 27(47.4%)cases,internal carotid artery occlusion in 24(42.1%)cases,and tandem lesions in 6(10.5%)cases.The time from onset to femoral artery puncture was 38.30(28.17,53.71)h,and the time from femoral artery puncture to vascular recanalization was 52.00(38.50,92.50)min.General anesthesia was the main anesthesia method,accounting for 64.9%(37/57).The etiological types of stroke were mainly large artery atherosclerosis(38 cases,66.7%),cardiogenic embolism(9 cases,15.8%),unknown causes(6 cases,10.5%),and other clear causes(4 cases,7.0%).Mechanical thrombectomy was the first choice in 41(71.9%)cases,balloon dilatation/stenting was used in 35(61.4%)cases,of which 15(26.3%)cases were the first choice.Finally,53(93.0%)cases were recanalized successfully.In terms of complications,1(1.8%)case had symptomatic intracranial hemorrhage.The 90-d prognosis rate was 59.6%(34/57),and 3(5.3%)cases died.Conclusion Endovascular treatment for AIS-LVO patients over 24 h after onset has high recanalization rate and good safety,but it still needs to be further verified by randomized controlled trials.

Objective To explore the effect of Sangxing Zhike Formula in rats with cough vari-ant asthma(CVA)and its possible mechanism based on the cyclic adenosine monophosphate(cAMP)/cystic fibrosis transmembrane conductance regulator(CFTR)pathway.Methods SD rats were randomly divided into control group,model group,dexamethasone group(0.5 mg/kg)and low-,medium-,high-dose Sangxing Zhike Formula groups(9.6,19.2 and 38.4 g/kg)using a ran-dom number table method,with 9 rats in each group.Except for the control group,CVA rat models were established in the other groups.Rats in each group were administered the drug by gavage once a day for 14 consecutive days.The general conditions of rats in each group were observed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in rat serum.Hematoxylin-eosin(HE)staining and periodic acid-Schiff(PAS)staining were used to observe the pathological changes in rat lung and bronchial tissues,and the acid-base balance of airway surface liquid(ASL)was measured.Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression levels of pro-tein kinase A(PKA),CFTR protein and their mRNA in lung tissues of rats.Results Compared with the control group,rats in the model group showed listlessness,dull fur,slow weight gain,a significantly expanded area of alveolar septal consolidation,and a large number of inflammatory cell infiltrations around the bronchi.Compared with the model group,rats in each intervention group had better general conditions and reduced inflammatory infiltrations in lung tissues and bronchial lu-mens.Compared with the control group,the serum levels of IL-1 β and IL-18 in the model group were increased,and the pH values of ASL in the model group,low-dose Sangxing Zhike Formula group,medium-dose Sangxing Zhike Formula group,and high-dose Sangxing Zhike Formula group were all decreased,with statistically significant differences(P＜0.05).Compared with the model group,the serum levels of IL-1β and IL-18 in each intervention group were decreased,and the pH values of ASL in the dexamethasone group,medium-dose Sangxing Zhike Formula group,and high-dose Sangxing Zhike Formula group were increased,with statistically significant differences(P＜0.05).Compared with the control group,the expressions of PKA protein and PKA mRNA in the model group,low-dose Sangxing Zhike Formula group,and medium-dose Sangxing Zhike Formula group were all decreased,and the expressions of CFTR protein and CFTR mRNA in the model group and each intervention group were all decreased,with statistically significant differences(P＜0.05).Compared with the model group,the expressions of PKA protein and PKA mRNA in the dexametha-sone group and high-dose Sangxing Zhike Formula group were increased,and the expressions of CFTR protein and CFTR mRNA in the high-dose Sangxing Zhike Formula group were increased,with statistically significant differences(P＜0.05).Conclusion Sangxing Zhike Formula can im-prove the general conditions of CVA rats,regulate the acid-base balance of ASL,reduce airway in-flammatory cell infiltration andairway remodeling,and decrease the levels of inflammatory factors IL-1β and IL-18.Its mechanism may be related to the cAMP/CFTR pathway.

BACKGROUND: Peripheral helper T (T PH ) cells are uniquely positioned within pathologically inflamed non-lymphoid tissues to stimulate B-cell responses and antibody production. However, the phenotype, function, and clinical relevance of T PH cells in hepatocellular carcinoma (HCC) are currently unknown. METHODS: Blood, tumor, and peritumoral liver tissue samples from 39 HCC patients (Sep 2016-Aug 2017) and 101 HCC patients (Sep 2011-Dec 2012) at the Third Affiliated Hospital of Sun Yat-sen University were used. Flow cytometry was used to quantify the expression, phenotype, and function of T PH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. T PH cells, CD19 + B cells, and T follicular helper (T FH ) cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. RESULTS: T PH cells highly infiltrated tumor tissues, which was correlated with tumor size, early recurrence, and shorter survival time. The tumor-infiltrated T PH cells showed a unique ICOS hi CXCL13 + IL-21 - MAF + BCL-6 - phenotype and triggered naïve B-cell differentiation into regulatory B cells. Triggering programmed cell death protein 1 (PD-1) induced the production of C-X-C motif chemokine ligand 13 (CXCL13) by T PH cells, which then suppressed tumor-specific immunity and promoted disease progression. CONCLUSION Our study reveals a novel regulatory mechanism of T PH cell-regulatory B-cell-mediated immunosuppression and provides an important perspective for determining the balance between the differentiation of protumorigenic T PH cells and that of antitumorigenic T FH cells in the HCC microenvironment.
Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Humans ; T-Lymphocytes, Helper-Inducer/metabolism* ; Animals ; Mice ; Male ; Female ; Mice, Inbred C57BL ; Middle Aged ; B-Lymphocytes, Regulatory/metabolism* ; Flow Cytometry ; Interleukin-21 ; Aged ; Chemokine CXCL13/metabolism*

Elemene is widely recognized as an effective anti-cancer compound and is routinely administered in Chinese clinical settings for the management of several solid tumors, including non-small cell lung cancer (NSCLC). However, its detailed molecular mechanism has not been adequately demonstrated. In this research, it was demonstrated that elemene effectively curtailed NSCLC growth in the patient-derived xenograft (PDX) model. Mechanistically, employing high-throughput screening techniques and subsequent biochemical validations such as microscale thermophoresis (MST), microRNA-145-5p (miR-145-5p) was pinpointed as a critical target through which elemene exerts its anti-tumor effects. Interestingly, elemene serves as a binding stabilizer for miR-145-5p, demonstrating a strong binding affinity (dissociation constant (K _D) = 0.39 ± 0.17 μg/mL) and preventing its degradation both in vitro and in vivo, while not interfering with the synthesis of the primary microRNA transcripts (pri-miRNAs) and precursor miRNAs (pre-miRNAs). The stabilization of miR-145-5p by elemene resulted in an increased level of this miRNA, subsequently suppressing NSCLC progression through the miR-145-5p/mitogen-activated protein kinase kinase kinase 3 (MAP3K3)/nuclear factor kappaB (NF-κB) pathway. Our findings provide a new perspective on revealing the interaction patterns between clinical anti-tumor drugs and miRNAs.

Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective To investigate the effect of intravitreal injection of fibrillin-2(FBN2)recombinant protein on FBN2-deficient retinopathy.Methods Thirty-two SPF-grade C57BL/6J mice were randomly divided into 4 groups:nor-mal control group,negative control group,FBN2 knockdown group,and FBN2 recombinant protein group,with 8 mice in each group.The right eyes were taken as the experimental eyes.Mice in the normal control group did not receive any inter-vention,mice in the negative control group were intravitreally injected with 3 μL empty vector(1 mg·L-1),and mice in the FBN2 knockdown group and FBN2 recombinant protein group were intravitreally injected with 3 μL adeno-associated vi-rus(1 mg·L-1).After 4 weeks,mice in the FBN2 recombinant protein group were intravitreally injected with 3 μL FBN2 recombinant protein(1 mg·L-1).Then,electroretinogram(ERG)and optical coherence tomography(OCT)were used to measure the amplitude of Rod-b and Max-a waves and the changes in the retinal structure.Real-time quantitative poly-merase chain reaction(RT-PCR)and Western blot were used to detect changes in FBN2,microfibril-associated glycopro-tein 2(MAGP-2),collagen I(COL1)mRNA and protein expression in the mouse retina.Results The ERG findings showed that compared with the negative control group and normal control group,the amplitude of Rod-b and Max-a waves in the retina of mice in the FBN2 knockdown group and FBN2 recombinant protein group decreased(all P＜0.05);com-pared with the FBN2 knockdown group,the amplitude of Rod-b and Max-a waves in the retina of mice in the FBN2 recom-binant protein group significantly increased(both P＜0.05).The OCT findings showed that compared with the FBN2 knock-down group,the structure of the retinal pigment epithelium and the light reflex in the FBN2 recombinant protein group be-came more regular.The RT-PCR detection results showed that compared with the FBN2 knockdown group,the expression of FBN2 mRNA in the retinal tissue of mice in the FBN2 recombinant protein group significantly increased,while the ex-pression of COL1 and MAGP-2 mRNA significantly decreased(all P＜0.05).Western blot assay results showed that com-pared with the FBN2 knockdown group,the expression of FBN2 protein in the retinal tissue of mice in the FBN2 recombi-nant protein group increased significantly,while the expression of COL1 and MAGP-2 proteins decreased significantly(all P＜0.05).Conclusion Intravitreal injection of FBN2 recombinant protein can compensate for the endogenous deficiency of FBN2 in mice with FBN2-deficient retinopathy and achieve therapeutic effects by regulating COL1 and MAGP-2 expres-sion.

Objective:To investigate the expression of latent transforming growth factor-β-binding protein (LTBP), transforming growth factor-β (TGF-β), cyclin-dependent kinase 2 (CDK2) and cyclin D2 (CCND2) in fibrillin-2 ( FBN2) interfering induced mouse retinopathy. Methods:Twenty-seven 8-week-old C57BL/6J mice were randomly divided into normal control group, empty vector group and FBN2 interference group according to the random number table method, with 9 mice in each group.The normal control group was not treated.The empty vector group and FBN2 interference group were intravitreally injected with 3 μl empty vector and 3 μl adeno-associated virus (AAV) carrying the sh-FBN2 interference plasmid in the right eye, respectively.The structural and functional changes of the retina were detected at 4 weeks after injection by optical coherence tomography (OCT) and full-field electroretinography (ERG).The expression and distribution of FBN2 protein in the retina were detected by immunofluorescence staining.The mRNA and protein expression levels of FBN2, LTBP-1, TGF-β2, CDK2 and CCND2 in mouse retina were detected by real-time fluorescence quantitative PCR and Western blot.All experiments complied with the ARVO statement.The research scheme was approved by the Experimental Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (No.2019036).Results:Four weeks after injection, the results of OCT examination showed that compared with normal control and empty vector groups, the retinal pigment cortex of the FBN2 interference group was irregular with high density reflection areas.Full-field ERG results showed that compared with normal control and empty vector groups, the amplitude of Rod-a, Rod-b, Max-a and Max-b waveforms in FBN2 interference group decreased, and the differences were statistically significant (all at P<0.05).The results of immunofluorescence staining showed that FBN2 was expressed in the whole retina, and the fluorescence intensity of FBN2 was weaker in FBN2 interference group than that in normal control and empty vector groups.The fluorescence intensity of FBN2 in normal control group, empty vector group and FBN2 interference group was 16.21±2.21, 15.57±3.63 and 5.32±1.06, respectively, with a statistically significant overall difference ( F=66.03, P<0.05).The fluorescence intensity of FBN2 protein in FBN2 interference group was significantly lower than that in empty carrier group and normal control group (both at P<0.05).Compared with normal control and empty vector groups, the relative expression levels of LTBP-1 and TGF-β2 mRNA and protein were significantly increased in FBN2 interference group, while the relative expression levels of FBN2, CDK2 and CCND2 mRNA and protein were significantly decreased, and the differences were statistically significant (all at P<0.05). Conclusions:The increase of LTBP-1 and TGF-β2 and the decrease of G1/S phase related proteins CDK2 and CCND2 are involved in the development of FBN2-deficient retinopathy.

BACKGROUND: Exosomes derived from breast cancer have been reported to play a role in promoting cell proliferation, migration, and angiogenesis, which has the potential to accelerate the healing process of diabetic wounds. The aim of this investigation was to examine the function of exosomes originating from 4T1 mouse breast carcinoma cells (TEXs) in the process of diabetic wound healing. METHODS: The assessment of primary mouse skin fibroblasts cell proliferation and migration was conducted through the utilization of CCK-8 and wound healing assays, while the tube formation of HUVECs was evaluated by tube formation assay. High-throughput sequencing, RT-qPCR and cell experiments were used to detect the roles of miR-126a-3p in HUVECs functions in vitro. The in vivo study employed a model of full-thickness excisional wounds in diabetic subjects to explore the potential therapeutic benefits of TEXs. Immunohistochemical and immunofluorescent techniques were utilized to evaluate histological changes in skin tissues. RESULTS: The findings suggested that TEXs facilitate diabetic wound healing through the activation of cell migration, proliferation, and angiogenesis. An upregulation of miR-126a-3p has been observed in TEXs, and it has demonstrated efficient transferability from 4T1 cells to HUVEC cells. The activation of the PI3K/Akt pathway has been attributed to miR-126a-3p derived from TEXs. CONCLUSIONS The promotion of chronic wound healing can be facilitated by TEXs through the activation of cellular migration, proliferation, and angiogenesis. The activation of the PI3K/Akt pathway by miR-126a-3p originating from TEXs has been discovered, indicating a potential avenue for enhancing the regenerative capabilities of wounds treated with TEXs.