Background Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated. Methods In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions. Results Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin. Conclusions These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.

Objective:To investigate the current situation of blood glucose monitoring in type 1 diabetes mellitus (T1DM) patients and its impact on glycemic control.Methods:The study was a cross-sectional survey. T1DM patients or their guardians who visited the Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University from October to December 2021 were selected as the research subjects. The patients′ age, course of diabetes, current treatment plan, education level, continuous glucose monitoring (CGM) and self-monitoring blood glucose (SMBG) were assessed by questionnaire. The glycated hemoglobin A 1c (HbA 1c) was measured. According to the Guidelines for the diagnosis and treatment of type 1 diabetes mellitus in China (2021 edition), HbA 1c<7.0% is defined as HbA 1c meeting the standard. Patients were divided into CGM group and SMBG group based on blood glucose monitoring methods. Mann-Whitney U test, chi-square test were used for inter-group comparison, and multivariate logistic regression analysis was used to analyze the influencing factors of HbA 1c compliance and different blood glucose monitoring methods. Results:A total of 197 patients with T1DM were included, with an average age of 27.0 (18.2, 41.0) years and a course of disease of 5.0 (2.0, 9.8) years. The utilization rates of CGM and SMBG were 61.4% (121/197) and 38.6% (76/197), respectively. The HbA 1c in the CGM group was lower than that in the SMBG group [6.6% (6.1%, 7.5%) and 7.5% (6.4%, 9.5%), Z=3.19, P<0.001], and the compliance rates of HbA 1c in the 2 groups were 62.1% (51/95) and 40.0% (24/60), respectively. The difference was statistically significant ( χ2=7.22, P<0.01). After adjustment for age, diabetes course and current treatment, logistic regression analysis showed that the use of CGM (OR=2.028, 95%CI 1.012-2.042, P<0.05) and education level (OR=1.571, 95%CI 1.209-2.042, P<0.01) were correlated with HbA 1c standard. Conclusion:In our real-world data, 61.4% of the patients with T1DM were using a CGM device, which was associated with improved glycemic control.

1型糖尿病(type 1 diabetes mellitus, T1DM)是一种由致病性CD4+、CD8+T细胞介导的胰岛β细胞进行性破坏的自身免疫性疾病[1].患者需要终身胰岛素治疗,尽管胰岛素替代疗法有助于改善糖代谢,但其不能完全替代内源性胰岛素的生物学功能,使患者面临低血糖发作和并发症的风险[2]. 胰岛移植(islet transplantation, IT)是目前根治T1DM的一种理想方式,从供体胰腺分离纯化得到的胰岛细胞经门静脉输注到受体肝脏内,从而建立内源性葡萄糖依赖的胰岛素分泌系统,恢复体内生理性胰岛素分泌模式,最终实现T1DM患者长期胰岛素独立性和血糖稳定.在过去的几十年,临床IT取得了长足的进步,特别是对于反复发生低血糖和低血糖意识障碍的患者[3],IT是有效方法.但IT作为治疗T1DM主要挑战是:胰岛来源有限、需要终身免疫抑制等 [4].

该文报道1例 GATA6基因杂合突变所致的青少年起病的糖尿病临床特征，并文献回顾 GATA6基因突变型糖尿病的病例特点。患者，男，16岁，诊断为1型糖尿病6年，伴有先天性心脏病病史，应用全外显子测序对患者进行分析，并用Sanger测序进行验证。基因测序结果显示，患者 GATA6基因4号外显子存在c.1367G>A杂合突变，患者父母均未发现相同的变异，为自发性突变。进一步影像学检查提示部分胰腺发育不全、胆囊缺失。患者使用胰岛素期间频发低血糖，胰岛素自身抗体阳性，考虑合并外源性胰岛素自身免疫综合征。上述结果为临床医师诊断青少年起病的糖尿病伴发多脏器受损提供思路，避免误诊、漏诊。

Objective:To explore whether palmitic acid (PA) can induce ferroptosis in pancreatic beta-cell line MIN6 cells.Methods:Pancreatic beta-cell line MIN6 cells were divided into normal group, PA group (1.0 mmol/L), PA+apoptosis inhibitor (Z-VAD-FMK) group (5 μmol/L), PA+ ferrostain-1(Fer-1) group (5 μmol/L), PA+ necrostatin-1(Nec-1) group (5 μmol/L). Each group was set three parallels. Cell viability was detected by CCK8 method. Cellular ultrastructure was assessed with electron microscopy. FerroOrange was used to detect intracellular Fe 2+. The lipid peroxidation assay kit was used to detect the concentration of malondialdehyde (MDA). Flow cytometry was used to detect intracellular reactive oxygen species (ROS) levels. Real-time quantitative polymerase chain reaction was used to detect the mRNA levels of caspase3, receptor-interacting protein kinase 3 ( RIPK3), acyl-CoA synthetase long-chain family member 4 ( ACSL4), prostaglandin endoperoxidase synthase 2 ( Ptgs2), arachidonate lipoxygenase 15 ( ALOX15), ferritin heavy chain ( FTH), ferritin light chain ( FTL), and glutathione peroxidase 4 ( GPX4). Western blotting was used to detect the proteins levels of cleaved caspase3, RIPK3, ACSL4, ALOX15 and Ptgs2. The t-test was used to compare two groups. Results:Compared with the PA group, cell viability of MIN6 was higher in PA+Z-VAD-FMK group, PA+Fer-1 group or PA+Nec-1 group ( P<0.01). MIN6 cells, which were treated with PA to exhibit the characteristic morphologic features, were associated with apoptosis, necrosis and ferroptosis under electron microscope. Compared with the control group, in PA group, the levels of the cellular intracellular Fe 2+, MDA and total lipid ROS were increased. The mRNA relative expression levels of caspase3, RIPK3, ACSL4, Ptgs2 and ALOX15 were increased. The mRNA relative expression levels of FTH and GPX4 were decreased. The protein relative expression levels of cleaved caspase3, RIPK3, ACSL4, ALOX15 and Ptgs2 were increased (all P<0.01). Compared with the PA group, in PA+Fer-1 group, the levels of cellular intracellular Fe 2+, MDA and total lipid ROS were decreased. The mRNA relative expression levels of ACSL4, Ptgs2 and ALOX15 were decreased. The mRNA relative expression levels of FTH and GPX4 were increased. The protein relative expression levels of ACSL4, ALOX15 and Ptgs2 were decreased (all P<0.01). Conclusion:PA could induce ferroptosis in pancreatic beta-cell line MIN6 cells.

Objective:To investigate the distribution of single nucleotide polymorphisms of interleukin-2 receptor α (IL2RA) gene in Chinese Han population and its correlation with classical type 1 diabetes (T1DM).Methods:A case-control study was conducted in T1DM patients from the First Affiliated Hospital of Nanjing Medical University and Children′s Hospital of Nanjing Medical University and healthy controls in this region from January 2008 to December 2014. The results of medical visits, such as serum C-peptide, zinc transporter 8 antibody (ZnT8A), glutamic acid decarboxylase antibody (GADA), protein tyrosine phosphatase antibody (IA-2A), and the genotyping of rs3118470 and rs2104286 in IL2RA gene were recorded. The t test, χ2 test and Kruskal-Wallis H test were used to compare the general information between T1DM patients and healthy controls and the clinical characteristics of different genotypes in T1DM patients. Logistic regression was performed to compare the frequency distributions of the alleles and genotypes between T1DM patients and controls, and the potential confounders ( e.g., age, sex) were adjusted. Univariate logistic regression was used to analyze the association between the polymorphism of IL2RA gene and T1DM susceptibility under different genetic models (dominant, recessive, super dominant and additive models). Results:The minor allele C of rs3118470 in IL2RA gene was the risk allele of T1DM ( P=0.026). The polymorphisms of rs3118470 in IL2RA gene were associated with T1DM susceptibility under dominant and additive genetic models [OR(95%CI)were 0.807 (0.666-0.978) and 0.880 (0.783-0.989), respectively, both P<0.05], but the clinical characteristics of T1DM patients were not statistically significantly different between different genotype groups (all P>0.05). There was a statistically significant difference in the positive rate of ZnT8A in T1DM patients among different genotypes of rs2104286 in IL2RA gene ( P=0.035). Conclusions:The polymorphisms of rs3118470 in IL2RA gene were significantly associated with the susceptibility of T1DM in Chinese Han population, and the minor allele C was the risk allele of T1DM. The polymorphisms of rs2104286 in IL2RA gene were correlated with the positive rate of ZnT8A.

目的:观察固肾泄浊和络方治疗糖尿病肾脏疾病的临床疗效及对患者血清中NF-κB和Ⅰ-κB水平的影响.方法:选取2016年09月至2018年06月诊治的DKDⅡ-Ⅳ期患者60例,随机分为对照组和治疗组,每组30例,两组均接受基础治疗,基础治疗主要控制血糖及血压,治疗组在基础治疗的基础上,服用固肾泄浊和络方(组成:生黄芪40g,党参15g,山萸肉10g,山药20g,白花蛇舌草20g,猫爪草30g,莪术15g,赤芍30g,土茯苓15g)[1],疗程均为2月,观察两组临床疗效及治疗前后HbAlc、MAlb、ACR、Scr、BUN、GFR、血清中NF-κB和Ⅰ-κB表达水平变化.结果:对照组和治疗组总有效率分别为66.67％和86.67％,治疗组总有效率较对照组显著提高,差异有统计学意义(P＜0.05).与治疗前比较,两组治疗后NF-κB和Ⅰ-κB水平明显降低,且治疗组上述指标降低程度优于对照组(P＜0.05).结论:固肾泄浊和络方治疗Ⅱ-Ⅳ期糖尿病肾病患者疗效显著,其机制与抑制NF-κB炎症信号通路的活化有关.