第一部分 原发性胆汁性胆管炎生物标志物的研究及抗线粒体抗体M2亚型阳性人群发生原发性胆汁性胆管炎的风险预测研究

研究内容Ⅰ. 原发性胆汁性胆管炎患者的血浆蛋白质组学研究

目的：原发性胆汁性胆管炎（Primary biliary cholangitis，PBC）是一种以肝内胆管进行性损伤、非化脓性胆管炎以及肝内胆汁淤积为主要特征的自身免疫性肝脏疾病。临床上诊断PBC须同时满足血清碱性磷酸酶或谷氨酰转肽酶异常升高，以及抗线粒体抗体（Anti-mitochondrial antibody，AMA）或AMA-M2阳性。然而，部分PBC患者不能同时满足以上条件，所以需要挖掘用于PBC诊断的新型标志物。熊脱氧胆酸（Ursodeoxycholic acid，UDCA）是治疗PBC的一线药物，然而临床上约有40%的PBC患者对UDCA治疗不应答，早期发现UDCA治疗无效的PBC患者有助于尽早应用二线治疗方案改善其预后。本研究基于PBC患者的蛋白质组学分析，筛选可用于PBC患者诊断及反映UDCA治疗效果的蛋白质类生物标志物。

方法：基于数据非依赖型采集质谱技术联合抗体芯片技术构建的高通量蛋白质组学检测平台，针对51例PBC患者与15例健康对照（Healthy controls，HCs）的血浆样本开展蛋白质组学分析。挖掘两组间差异蛋白，对其进行生物信息学分析、蛋白表达趋势分析、药物靶点分析以及与反映PBC活动性指标的相关性分析，从而发现具有PBC诊断及反映UDCA治疗效果的差异蛋白，并通过酶联免疫吸附试验（Enzyme linked immunosorbent assay，ELISA）在包含129例PBC患者、16例干燥综合征患者、16例慢性乙型肝炎患者、8例自身免疫性肝炎患者以及31例HCs的独立验证队列中，对候选差异蛋白作为PBC标志物的性能进行分析。

结果：PBC患者与HCs之间，基于高通量蛋白质组学检测平台共筛选到361个差异蛋白。生物信息学分析结果显示，361个差异蛋白参与5个主要生物学过程类别，分别为免疫反应、细胞迁移、细胞间粘附作用、淋巴细胞活化调节以及细胞外调节蛋白激酶级联传导；通路富集分析显示，上调差异蛋白主要富集于细胞因子受体相互作用通路、病毒蛋白与细胞因子及其受体相互作用通路等炎症调控相关通路，下调蛋白主要富集于氮素代谢、维生素消化吸收等代谢相关通路。基于PBC患者的UDCA治疗状态进行差异蛋白表达趋势分析，可将361个差异蛋白分为4个聚类并进行通路富集分析，发现NF-kappa B信号通路中富集了多个蛋白在UDCA治疗不应答PBC患者中的表达水平显著高于UDCA治疗应答PBC患者。药物靶点分析结果显示，91个差异蛋白是疾病药物治疗的潜在靶点，其中13个差异蛋白可作为治疗自身免疫病的药物靶点，11个差异蛋白可作为治疗消化系统肿瘤的药物靶点。相关性分析显示，26个差异蛋白可同时与5种反映PBC疾病活动性的实验室检查指标具有显著相关性。最终选取了10个候选差异蛋白进行ELISA验证，发现6个差异蛋白即多聚免疫球蛋白受体、细胞间黏附分子1、膜内氨酰氨基肽酶、血管细胞黏附因子1、转化生长因子-β诱导蛋白IG-H3（Transforming growth factor-beta-induced protein ig-h3，TGFBI）以及CD163，均可作为PBC患者诊断及反映UDCA治疗效果的蛋白质类标志物，其中TGFBI从未在PBC中进行报道，该蛋白在PBC诊断及区分UDCA治疗应答与不应答PBC患者的曲线下面积值均为0.824。

结论：本研究利用高通量蛋白质组学检测平台描绘了PBC患者血浆蛋白质组学特征，揭示了PBC患者不同的UDCA治疗状态对其血浆蛋白质表达谱的影响，验证了6个具有PBC诊断及反映UDCA治疗反应性标志物潜力的差异蛋白，其中TGFBI在PBC中首次报道，该蛋白在PBC发病机制中的作用值得进一步探索。

研究内容Ⅱ.原发性胆汁性胆管炎患者的血浆脂质组学研究

目的：脂质代谢异常在原发性胆汁性胆管炎（Primary biliary cholangitis，PBC）患者中普遍存在。干燥综合征（Sjögren’s syndrome，SS）是PBC患者最容易合并的自身免疫性肝外疾病，临床上大约有35%的PBC患者可合并SS。然而，这两种疾病的脂质特征尚未完全明确。因此，本研究通过对两种疾病的血浆脂质类代谢物变化进行分析，揭示两种疾病的血浆脂质代谢特征，筛选可用于PBC诊断及反映熊脱氧胆酸（Ursodeoxycholic acid，UDCA）治疗效果的脂质类标志物。

方法：基于超高效液相色谱联用高分辨质谱技术针对60例PBC患者、30例SS患者以及30例健康对照（Healthy control，HCs）的血浆样本进行非靶向脂质组学分析。通过正交偏最小二乘判别分析筛选PBC患者与HCs之间的、PBC患者与SS之间的差异脂质进行筛选，采用无监督的一致性聚类分析对PBC患者进行基于脂质组学数据的分型，利用相关性分析筛选可以反映PBC疾病活动性的差异脂质，受试者工作曲线（Receiver operating characteristic，ROC）分析评价差异脂质作为PBC生物标志物的潜能。进一步通过酶联免疫吸附试验（Enzyme linked immunosorbent assay，ELISA）在包含48例PBC患者、16例SS患者、16例HCs的独立队列中，对候选差异脂质进行验证。

结果：PBC患者与HCs之间，共发现115个差异脂质且在PBC患者血浆中均上调表达，其差异脂质的种类为磷脂酰胆碱（Phosphatidylcholine，PC）、甘油三酯（triacylglycerol，TG）、磷酸乙醇胺（Phosphoethanolamine，PE）、甘油二酯（Diacylglycerol，DG）、磷酸肌醇（Phosphoinositol，PI）、神经酰胺（Ceramide，Cer），溶血磷脂酰胆碱（Lysophosphatidylcholines，LysoPC），胆固醇或胆固醇酯、鞘磷脂（Sphingomyelin，SM）以及葡萄糖神经酰胺。一致性聚类分析显示，基于PBC患者的脂质组学数据可将其分为三个亚型，其中亚型1中PBC患者的疾病严重程度高于亚型2与亚型3中的PBC患者，UDCA治疗不应答PBC患者的占比最高。相关性分析显示，17个脂质可同时与5个反映PBC疾病活性的实验室检查指标呈显著正相关，ROC分析表明17个脂质均可显著区分UDCA治疗应答与UDCA治疗不应答的PBC患者，其曲线下面积（Area under curve，AUC）值从大到小排名前6且AUC值大于0.9的脂质分别为PC(16:0/16:0)、PC(18:1/8:1)、PC(42:2)、PC(16:0/18:1)、PC(17:1/14:0)以及PC(15:0/18:1)。PBC患者与HCs之间，共发现44个差异脂质且在PBC患者血浆中均上调表达，其差异脂质的种类为PC、酰基肉碱、PE、LysoPC、PI、TG、DG、Cer以及SM。将HCs与SS患者作为ROC分析的对照组，其中8个脂质诊断PBC的AUC值大于0.9，分别为LysoPC(16:1)、PC(16:0/16:0)、PC(16:0/16:1)、PC(16:1/20:4)、PC(18:0/20:3)、PC(18:1/20:2)、PC(20:0/22:5)以及PC(20:1/22:5)。ELISA验证结果显示PC在PBC患者及SS患者血浆中的表达水平与HCs相比显著升高（P = 0.005；P = 0.012），且在UDCA治疗不应答PBC患者血浆中的表达水平显著高于UDCA治疗应答PBC患者（P = 0.028）。ROC分析显示PC可将PBC患者从HCs与SS患者中显著区分，其AUC值为0.639（P = 0.037）；亦可显著区分UDCA治疗应答与UDCA治疗不应答的PBC患者，其AUC值为0.794（P = 0.019）。

结论：本研究揭示了PBC患者与HCs及SS患者之间血浆脂质组特征。磷脂代谢紊乱可能是PBC患者与SS患者脂质代谢异常的共同特征。PC是与PBC患者的疾病活动性和UDCA治疗反应性相关的主要脂质种类。

研究内容Ⅲ. 原发性胆汁性胆管炎患者外周血固有淋巴细胞的表达及其临床意义

目的：固有淋巴细胞（Innate lymphoid cells，ILCs）是一组新型的先天免疫细胞，可通过对组织驻留细胞表达的免疫信号进行快速响应从而在早期免疫反应中发挥关键作用。ILCs参与多种自身免疫性疾病的发病机制。然而，ILCs在原发性胆汁性胆管炎（primary biliary cholangitis，PBC）患者中的表达情况尚不明确。因此，本研究旨在调查ILCs在PBC患者外周血中的表达情况及其临床意义。

方法：本研究针对48例PBC患者及24例健康对照（Healthy controls，HCs）外周全血样本中ILCs及其亚群的表达情况利用多色流式细胞术进行检测，并分析了活化标记CD38、CD69及CD45RO在ILCs亚群中的表达情况以及总ILCs中细胞因子干扰素γ、白介素17A（Interleukin 17A，IL17A）及白介素13的表达水平。

结果：PBC患者外周血中总ILCs、ILC前体（ILC precursors，ILCPs）和ILCP/ILC2比值的水平与HCs相比显著增加，ILC2s在PBC患者外周血中的表达水平显著降低，ILCPs、ILCP/ILC2比值在PBC患者外周血中的水平显著上升。PBC患者外周血中ILC2s的表达水平与谷氨酰转肽酶（Gamma-glutamyl transpeptidase，GGT）、谷丙转氨酶（Alanine aminotransferase，ALT）、谷草转氨酶（Aspartate aminotransferase，AST）的表达水平呈显著负相关，ILCPs、ILCP/ILC2比值在PBC患者中的水平与其碱性磷酸酶、GGT、ALT及AST水平呈显著正相关。ILC2s在熊脱氧胆酸（ursodeoxycholic acid，UDCA）治疗不应答PBC患者中的表达水平显著低于UDCA治疗应答PBC患者，而ILCPs和ILCP/ILC2比值水平在UDCA治疗不应答PBC患者中的表达水平显著增加。PBC患者外周血中CD38⁺ILC2s、CD38⁺ILCPs、CD45RO⁺ILC2s及CD45RO⁺ILCPs的表达水平显著高于HCs。PBC患者外周血中分泌IL17A的ILCs水平显著高于HCs。

结论：PBC患者外周血中ILCs及其亚群的水平发生改变。PBC患者外周血ILC2s、ILCPs以及ILCP/ILC2比值水平与PBC患者的疾病活动性密切相关。PBC患者外周血ILC2s、ILCPs以及ILCP/ILC2比值水平的改变可能反映了PBC患者UDCA治疗应答效果不佳。PBC患者外周血中ILC2s与ILCPs发生活化，这些活化的ILCs可能参与PBC的发病机制。

研究内容Ⅳ. 抗线粒体抗体M2亚型阳性健康体检人群发生原发性胆汁性胆管炎的风险预测研究

目的：抗线粒体抗体M2亚型（Anti-mitochondrial M2 antibody，AMA-M2）是诊断原发性胆汁性胆管炎（Primary biliary cholangitis，PBC）的特异性标志物，AMA-M2也可存在于未患有PBC的健康人群或其它疾病的患者中。本研究旨在调查AMA-M2在中国健康体检人群中的流行情况，探究未患有PBC的AMA-M2阳性人群发生PBC的风险因素并构建该人群发展为PBC的风险预测模型。

方法：2010年至2022年期间，共计有72173名体检者曾在北京协和医院体检时检测过AMA-M2，根据体检结果筛选未患有PBC的AMA-M2阳性人群。针对该类人群进行随访，观察其是否在随访期间发展为PBC，并收集其基线临床特征及实验室检查数据。利用最小绝对收缩和选择运算符（Least absolute shrinkage and selection operator，LASSO）回归来筛选未患有PBC的AMA-M2阳性人群PBC发生的风险因素，使用多因素Logistic回归分析构建该人群随访期间发展为PBC的风险预测模型，继而利用受试者工作曲线（Receiver operating characteristic curve，ROC）分析对预测模型在训练集与验证集队列中的预测性能进行评价。

结果：共计有2333名体检者其AMA-M2抗体检测结果为阳性，阳性率约为3.23%；其中男性体检者939名，占所有男性体检者的2.49%；其中女性体检者1394名，占所有女性体检者的4.04%。此外，2333名AMA-M2阳性的体检者中，82名体检者在体检时已患有PBC，2076名体检者未患有PBC。针对2076名AMA-M2阳性体检者进行随访，经中位时间6.6年的随访（四分位距：4.8-8.1），13例基线时未患有PBC的AMA-M2阳性体检者随访期间发展为PBC，其5年累积发病率约为0.5%（95% CI: 0.2-0.9）。LASSO回归分析显示碱性磷酸酶（Alkaline phosphatase，ALP）、谷氨酰转肽酶（Glutamyl transpeptidase，GGT）、免疫球蛋白M（Immunoglobulin M，IgM）、嗜酸性粒细胞比例（Eosinophils percentage，EOS%）、γ球蛋白百分比以及血红蛋白（Hemoglobin，Hb）是PBC发生密切相关的最佳预测因子。多因素Logistic回归分析显示，在基线时未患有PBC的AMA-M2阳性个体中，所纳入的预测因子ALP（OR = 1.047，95% CI: 1.034-1.061）、GGT（OR = 1.014，95% CI: 1.009-1.019）、IgM（OR = 1.615，95% CI: 1.170-2.291）、ESO%（OR = 1.211，95% CI: 1.056-1.379）以及γ球蛋白百分比（OR = 1.150，95% CI: 1.022-1.284）为AMA-M2阳性体检人群发展为PBC的独立预测风险因素。多因素Logistic回归基于LASSO回归筛选的6个预测因子构建预测模型，ROC分析显示训练集、验证集1及验证集2队列中该预测模型曲线下面积值分别为0.954（95% CI: 0.933-0.976）、0.975（95% CI: 0.953-0.998）以及0.917（95% CI: 0.847-0.987）。

结论：未患有PBC的AMA-M2阳性体检人群经随访后发现，其新发PBC的累积发病率较低。基于ALP、GGT、IgM、EOS%、γ球蛋白百分比以及Hb这6个指标构成的预测模型对于未患有PBC的AMA-M2阳性体检人群其PBC发生风险具有一定的预测能力，可为早期识别PBC的高风险人群提供参考依据。

第二部分 原发性胆汁性胆管炎患者与糖尿病患者接种新型冠状病毒疫苗安全性和加强接种免疫原性研究

研究内容Ⅴ. 原发性胆汁性胆管炎患者接种新冠灭活疫苗的安全性及加强接种免疫原性研究

目的：新冠疫苗对于控制严重急性呼吸综合征冠状病毒2（Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）的流行及降低SARS-CoV-2引发的新型冠状病毒肺炎（Coronavirus disease 2019，COVID-19）的重症率及死亡率具有重要作用。灭活疫苗是中国地区接种人数最多的新冠疫苗类型。原发性胆汁性胆管炎（Primary biliary cholangitis，PBC）是一种以肝内小胆管非化脓性炎症及血清中可检测出抗线粒体抗体为主要特征的自身免疫性疾病，PBC的发生可导致机体免疫系统功能失调。PBC患者接种新冠灭活疫苗的安全性及免疫原性尚不明确。因此，本研究针对PBC患者接种新冠灭活疫苗后的安全性与免疫原性开展调查。

方法：本研究构建了两个研究队列，一个研究队列纳入了73例PBC患者与73例健康对照（Healthy controls，HCs）用于调查PBC患者接种新冠灭活疫苗后的抗体反应来反映其免疫原性，另一个研究队列纳入了86例PBC患者通过填写在线调查问卷探究其实际接种情况、对接种新冠疫苗的态度以及接种安全性。通过检测抗受体结合域（Receptor binding domain，RBD）特异性IgG型抗体水平、SARS-CoV-2野生型中和抗体抑制率与 SARS-CoV-2奥密克戎BA.4/5毒株中和抗体抑制率来评价PBC患者接种新冠灭活疫苗诱导的体液免疫应答反应。

结果：加强免疫接种新冠灭活疫苗6个月后PBC患者抗RBD特异性IgG型抗体与SARS-CoV-2野生型中和抗体抑制率与HCs相比显著减低。PBC患者与HCs加强免疫接种后其SARS-CoV-2奥密克戎BA.4/5 毒株中和抗体抑制率水平仍低于最小有效抑制率。PBC患者的实验室检查指标未发现与任何SARS-CoV-2抗体具有显著相关性。86例接受在线调查问卷的PBC患者中，仅有21例PBC患者完成加强免疫接种新冠疫苗，另有53例PBC患者从未接种过新冠疫苗。未接种新冠疫苗的PBC患者与接种者相比更加担忧新冠疫苗安全性，尚未接种新冠疫苗的主要原因为“医生不推荐接种新冠疫苗”。新冠灭活疫苗第一剂、第二剂以及第三剂接种在参与在线问卷调查PBC患者中的不良反应率分别为6.1%、10.3%以及9.5%，没有PBC患者报告接种新冠灭活疫苗后出现威胁生命或需要住院治疗的严重不良反应。

结论：PBC患者加强免疫接种新冠灭活疫苗6个月后其疫苗诱导的抗体应答反应与HCs相比显著降低。PBC患者与HCs加强免疫接种新冠灭活疫苗后仍具有奥密克戎变异体突破性感染的风险。PBC患者接种新冠灭活疫苗是相对安全的，但仍需在更大样本量的PBC患者队列中进行验证。

研究内容Ⅵ. 新冠疫苗在中国糖尿病患者中的接种情况、接种态度及其安全性调查

目的：糖尿病（Diabetes mellitus，DM）是我国最常见的慢性疾病之一，该病是严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）感染和新型冠状病毒肺炎预后不良的危险因素。新冠疫苗是控制SARS-CoV-2大流行的关键措施之一。然而，中国DM患者新冠疫苗接种的实际覆盖率及其影响接种行为的相关因素尚不明确。因此，本研究旨在调查中国DM患者新冠疫苗的实际接种率、接种态度以及安全性。

方法：利用问卷星平台设计线上调查问卷。在2022年8月3日至2022年10月3日期间，针对来自中国范围内180家三级医院就诊于内分泌科的DM患者开展基于横断面研究设计的问卷调查研究，收集其接种新冠疫苗的具体情况、接种后的不良反应情况以及对新冠疫苗态度等信息。采用多元Logistic回归分析模型探究影响DM患者新冠疫苗接种行为的潜在因素。

结果：本研究共收集到2200例DM患者的有效问卷，结果显示有1929例DM患者（87.7%）至少接种了一剂新冠疫苗，271例DM患者（12.3%）在参与调查前从未接种过新冠疫苗。此外，65.2%的DM患者（n = 1434）已完成新冠疫苗加强免疫接种，而16.2%的DM患者（n = 357）仅进行了新冠疫苗完全接种，6.3%的DM患者（n = 138）仅完成了新冠疫苗的部分接种。新冠疫苗第一剂、第二剂以及第三剂接种在参与调查DM患者中的不良反应率分别为6.0%、6.0%以及4.3%。多元Logistic回归分析显示，DM患者合并免疫炎症性疾病[部分接种：比值比（Odds ratio，OR）= 0.12，95%置信区间（Confidence interval，CI）：0.02-0.99，P = 0.049；完全接种：OR = 0.11，95% CI: 0.02-0.51，P = 0.005；加强免疫接种：OR = 0.28，95% CI: 0.12-0.64，P = 0.002]、合并糖尿病肾病[部分接种：OR = 0.23，95% CI: 0.09-0.58，P = 0.048；完全接种：OR = 0.51，95% CI: 0.26-0.99，P = 0.048；加强免疫接种：OR = 0.30，95% CI: 0.17-0.53，P ＜ 0.001]以及对新冠疫苗安全性的认知[部分接种：OR = 0.44，95% CI: 0.22-0.87，P = 0.018；完全接种：OR = 0.48，95% CI: 0.28-0.82，P = 0.007；加强免疫接种：OR = 0.45，95% CI: 0.29-0.70，P ＜ 0.001]是影响三种新冠疫苗接种行为的相关因素。

结论：本研究调查显示，中国DM患者新冠疫苗实际接种率较高。DM患者的合并疾病情况以及对新冠疫苗安全性的认知影响了DM患者新冠疫苗接种行为。本研究调查未观察到DM患者中接种新冠疫苗后发生严重不良反应事件，表明新冠疫苗接种对于DM患者是相对安全的。

研究内容Ⅶ. 2型糖尿病患者加强接种新冠灭活疫苗的免疫原性研究

目的：2型糖尿病（Type 2 diabetes mellitus，T2DM）患者是易于感染严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）且感染后容易发展为重症的高风险人群。灭活疫苗是在中国接种范围最广的新冠疫苗类型。然而，T2DM患者加强免疫接种新冠灭活疫苗后的免疫原性尚不明确。本研究旨在调查T2DM患者加强免疫接种新冠灭活疫苗后对新冠灭活疫苗的抗体反应。

方法：本研究共纳入已接种新冠灭活疫苗的201例T2DM患者和102例健康对照组（Healthy controls，HCs）。通过检测抗SARS-CoV-2总抗体滴度、抗受体结合域（Receptor binding domain，RBD）特异性IgG型抗体水平、SARS-CoV-2野生型中和抗体抑制率与SARS-CoV-2奥密克戎BA.4/5毒株中和抗体抑制率来评价T2DM患者接种新冠灭活疫苗诱导的体液免疫应答反应。

结果：加强免疫接种新冠灭活疫苗后6个月内，抗SARS-CoV-2总抗体、抗RBD特异性IgG型抗体、SARS-CoV-2野生型中和抗体与SARS-CoV-2奥密克戎BA.4/5毒株中和抗体水平在T2DM患者与HCs之间均无明显变化。然而，加强免疫接种新冠灭活疫苗6个月以后，T2DM患者的抗RBD特异性IgG型抗体水平（P = 0.018）和SARS-CoV-2野生型中和抗体抑制率（P = 0.007）与HCs相比显著减低。T2DM患者与HCs加强免疫接种新冠灭活疫苗后，由于SARS-CoV-2奥密克戎BA.4/5毒株中和抗体抑制率水平仍低于最小有效抑制率，导致其仍无法抵御奥密克戎BA.4/5毒株感染。T2DM患者加强免疫接种新冠灭活疫苗后其抗RBD特异性IgG型抗体水平与其外周血CD3⁺CD4^-CD8^-T细胞比例呈显著正相关（P = 0.045），并且加强免疫后抗RBD特异性IgG型抗体阳性的T2DM患者其外周血CD3⁺CD4^-CD8^-T细胞比例水平显著高于抗RBD特异性IgG型抗体阴性的T2DM患者（P = 0.005）。

结论：加强免疫接种新冠灭活疫苗6个月以内，T2DM患者与HCs对于疫苗诱导的抗体反应模式相似，与HCs相比其抗体应答反应无明显差异。然而，T2DM患者在加强免疫接种新冠灭活疫苗超过6个月后与HCs相比其体液免疫应答反应受损。此外，T2DM患者与HCs加强免疫接种新冠灭活疫苗后仍具有奥密克戎变异体突破性感染的风险。

Part I: Research on Biomarkers of Primary Biliary Cholangitis and Risk Prediction of Primary Biliary Cholangitis in Individuals with Positive Anti-mitochondrial Antibody M2 Subtype

Project I. Plasma Proteomic Study in Patients with Primary Biliary Cholangitis

Objective: Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by progressive intrahepatic bile duct injury, non-suppurative cholangitis, and intrahepatic cholestasis. Clinically, PBC must simultaneously satisfy abnormal alkaline phosphatase  or glutamyl transpeptidase elevation. And positive for anti-mitochondrial antibody (AMA) or AMA-M2. However, some patients with PBC do not meet the above conditions at the same time, so it is necessary to explore new biomarkers for PBC diagnosis. Ursodeoxycholic acid (UDCA) is a first-line medicine for the treatment of PBC. However, about 40% of patients with PBC do not respond to UDCA treatment. Early discovery of patients with PBC who do not respond to UDCA treatment is conducive to early application of second-line treatment to improve their prognosis. Based on proteomic analysis of PBC patients, this study screened protein biomarkers that could be used to diagnose patients with PBC and reflect the therapeutic effect of UDCA.Methods: The plasma samples of 51 patients with PBC and 15 healthy controls (HCs) were analyzed by a high throughput proteomic detection platform based on data-independent acquisition mass spectrometry combined with antibody microarray technology. The differential proteins between the two groups were mined, and bioinformatics analysis, protein expression trend analysis, drug target analysis, and correlation analysis with PBC activity markers were performed to find the differential proteins that can diagnose PBC and reflect the therapeutic effect of UDCA. In addition, enzyme linked immunosorbent assay (ELISA) was performed on 129 patients with PBC, 16 patients with Sjogren's syndrome, 16 patients with chronic hepatitis B, 8 patients with autoimmune hepatitis, and 31 HCs in the independent validation cohort, in order to analysis the performance of candidate differential proteins as PBC markers.

Results: A total of 361 different proteins were identified between patients with and HCs based on a high-throughput proteomic detection platform. The results of bioinformatics analysis showed that 361 differential proteins were involved in five major biological processes, namely immune response, cell migration, intercellular adhesion, lymphocyte activation regulation, and extracellular regulatory protein kinase cascade conduction. Pathway enrichment analysis showed that upregulated differential proteins were mainly concentrated in Cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor related to inflammation regulation, and down-regulated proteins were mainly concentrated in nitrogen metabolism, vitamin digestion and absorption, and other metabolic pathways. Based on the UDCA treatment response of patients with PBC, the expression trend of differential proteins was analyzed. Three hundred and sixty-one differential proteins could be divided into 4 clusters and pathway enrichment analysis was performed. It was observed that many proteins were enriched in the NF-kappa B signaling pathway, and their expression level showed an upward trend from UDCA-responder to UDCA-non-responder. The results of drug target analysis showed that 91 differential proteins were potential targets for therapy, 13 differential proteins could be used as targets for autoimmune diseases, and 11 differential proteins could be used as targets for digestive system tumors. Correlation analysis showed that 26 differential proteins were significantly correlated with 5 laboratory indicators of PBC disease activity at the same time. Finally, 10 candidate differential proteins were chosen for ELISA validation. Six differential proteins were validated, which were poly immunoglobulin receptor, intercellular adhesion molecule 1, alanyl aminopeptidase, vascular cell adhesion molecule 1, transforming growth factor-beta-induced protein IG-H3 (TGFBI), and CD163. The above six proteins can be used as protein biomarkers for the diagnosis of patients with PBC and reflect the therapeutic effect of UDCA. Among them, TGFBI has never been reported in PBC, and the area under the curve for the diagnosis of PBC and the difference between UDCA-responder and UDCA-non-responder is 0.824.

Conclusion: In this study, a high-throughput proteomic detection platform was used to describe the plasma proteomic characteristics of PBC patients, revealing the influence of different UDCA treatment states on the plasma protein expression profile of PBC patients, and confirming 6 differential proteins that have the potential to diagnose PBC and reflect the therapeutic responsiveness markers of UDCA. TGFBI was first reported in PBC in this study. The role of this protein in the pathogenesis of PBC deserves further exploration.

Project Ⅱ. Plasma Lipidomic Study in Patients with Primary Biliary Cholangitis

Objective: Abnormal lipid metabolism is common in all patients with primary biliary cholangitis (PBC). Sjogren's syndrome (SS) is the most common autoimmune extrahepatic disease in patients with PBC, and about 35% of patients with PBC could be complicated with SS. However, the lipid profile of these two diseases has not been fully defined. Therefore, by analyzing the alternations of plasma lipid metabolites in the two diseases, this study revealed the characteristics of plasma lipid metabolism in the two diseases, and screened lipid biomarkers that could be used in the diagnosis of PBC and reflect the therapeutic effect of Ursodeoxycholic acid (UDCA).

Methods: Plasma samples from 60 patients with PBC, 30 patients with SS, and 30 healthy controls (HCs) were analyzed by non-targeted lipidomics based on ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry. Differential lipids between patients with PBC and HCs and between patients with PBC and SS were screened by orthogonal partial least squares discriminant analysis. Unsupervised consistent cluster analysis was used to classify patients with PBC based on lipidomic data. Differential lipids reflecting the disease activity of PBC were screened by correlation analysis. The potential of differential lipids as biomarkers of PBC was evaluated by receiver operating characteristic (ROC) analysis. The candidate differential lipids were further validated by enzyme linked immunosorbent assay (ELISA) in an independent cohort of 48 patients with PBC, 16 patients with SS, and 16 patients with HCs.

Results: A total of 115 differential lipids were found between PBC patients and HC patients, and all of them were up-regulated in the plasma of PBC patients. The differential lipids included phosphatidylcholine (PC) and triacylglycerol (TG), phosphoethanolamine (PE), diacylglycerol (DG), phosphoinositol (PI), ceramide (Cer), lysophosphatidylcholines (LysoPC), cholesterol or cholesterol esters, sphingomyelins (SM), and glucosylceramide. Unsupervised consensus clustering showed that PBC patients could be divided into three subtypes based on lipidomics data, among which patients with PBC in subtype 1 had higher disease severity than PBC patients in subtype 2 and subtype 3, and the proportion of patients with PBC who did not respond to UDCA treatment was the highest. Correlation analysis showed that 17 lipids were significantly positively associated with 5 laboratory test indicators reflecting PBC disease activity at the same time. ROC analysis showed that all 17 lipids could significantly distinguish PBC patients who responded to UDCA treatment from those who did not respond to UDCA treatment and the top 6 lipids with area under curve (AUC)  greater than 0.9 from largest to smallest were PC(16:0/16:0), PC(18:1/8:1), PC(42:2), PC(16:0/18:1), PC(17:1/14:0) and PC(15:0/18:1). A total of 44 differential lipids were found between PBC patients and HCs, all of which were up-regulated in the plasma of PBC patients, and the types of differential lipids were PC, acylcarnitines, PE, LysoPC, PI, TG, DG, Cer and SM. Patients with SS and HCs were used as the control group for ROC analysis, and the AUC value of 8 lipid-diagnosed PBC was greater than 0.9, which is LysoPC(16:1), PC(16:0/16:0), PC(16:0/16:1), PC(16:1/20:4), PC(18:0/20:3), PC(18:1/20:2), PC(20:0/22:5) and PC(20:1/22:5), respectively. ELISA detection showed that the expression level of PC in the plasma of patients with PBC and SS was significantly higher than that of HCs (P = 0.005; P = 0.012), and the expression level in the plasma of patients with PBC who did not respond to UDCA treatment was significantly higher than that of PBC patients who responded to UDCA treatment (P = 0.028). ROC analysis showed that PC could significantly distinguish patients with PBC from HCs and SS, with an AUC of 0.639 (P = 0.037); patients with PBC who responded to UDCA treatment and those who did not respond to UDCA treatment can also be significantly distinguished, with an AUC of 0.794 (P=0.019).

Conclusion: This study revealed the characteristics of plasma lipids between patients with PBC, and HCs and patients with SS. Dysregulation of phospholipid metabolism may be a common feature of dysregulation of lipid metabolism in patients with PBC and patients with SS. PC is the major lipid class associated with disease activity and UDCA treatment response in patients with PBC.

Project Ⅲ. Expression of Innate Lymphoid Cells in Peripheral Blood of Patients with Primary Biliary Cholangitis and Its Clinical Significance

Objective: Innate lymphoid cells (ILCs), a novel group of innate immune cells, play a key role in the early immune response via rapidly reacting to signals expressed by tissue-resident cells. ILCs contribute to some autoimmune diseases. The expression of ILCs in patients with primary biliary cholangitis (PBC) is not well defined. We aim to investigate the proportions of circulating ILC subgroups in patients with PBC and its clinical significance.

Methods: Overall, 48 patients with PBC and 24 healthy controls (HCs) were enrolled. Circulating ILCs were detected by flow cytometry. Furthermore, the expression of activation markers CD38, CD69, and CD45RO in ILCs subgroups and the levels of cytokines interferon-γ, interleukin 17A (IL17A), and interleukin-13 in total ILCs were analyzed.

Results: The proportions of total ILCs, ILC precursors (ILCPs), and ILCP/ILC2 ratio increased and that of ILC2s decreased in patients with PBC. ILC2 proportion was negatively correlated with gamma-glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). The proportion of ILCPs and ILCP/ILC2 ratio were positively correlated with alkaline phosphatase, GGT, ALT, and AST. ILC2 proportion was significantly decreased in the ursodeoxycholic acid (UDCA) -non-responder group compared with the UDCA-responder group, whereas the proportion of ILCPs and ILCP/ILC2 ratio were significantly increased. The proportions of CD38⁺ILC2s, CD38⁺ILCPs, CD45RO⁺ILC2s, and CD45RO⁺ILCPs were significantly higher in patients with PBC than in HCs. Levels of IL17A-producing ILCs were higher in patients with PBC than in HCs. PBC is accompanied by alterations in circulating ILCs.

Conclusion: The proportions of ILC2s, ILCPs, and ILCP/ILC2 ratio were associated with the PBC disease activity. The proportions of ILCPs and ILCP/ILC2 ratio may reflect the UDCA treatment failure in patients with PBC. ILC2s and ILCPs from patients with PBC get activated, these cells may be involved in the pathogenesis of PBC.

Project Ⅳ. Prediction of New-onset Primary Biliary Cholangitis (PBC) among Health Check-up Population with Anti-mitochondrial M2 Antibody Positive but without Baseline PBC

Objective: Anti-mitochondrial M2 antibody (AMA-M2) is a specific biomarker for primary biliary cholangitis (PBC) and it could be also presented in non-PBC individuals. We aim to estimate the prevalence of AMA-M2 in the health check-up population and investigate the incidence of PBC and predictors for PBC development in AMA-M2-positive individuals without PBC at baseline.

Methods: A total of 72173 health check-up individuals tested AMA-M2 in Peking Union Medical College Hospital from 2010 to 2022, of which non-PBC AMA-M2 positive individuals were performed follow-up. Baseline data of both clinical characteristics and laboratory examinations were collected in all AMA-M2-positive individuals. Least absolute shrinkage and selection operator (LASSO) regression was performed to investigate the predictors for developing PBC. Multivariate logistic regression analysis was used to construct a risk prediction model for PBC in this population during the follow-up period. The receiver operating characteristic curve (ROC) analysis was used to evaluate the prediction performance of the risk prediction model in the training set and validation set cohorts.

Results: A total of 2333 individuals (about 3.23% of the 72173 individuals) were detected with AMA-M2, including 939 males (2.49%) and 1394 females (4.04%). Eighty-two individuals (3.8%) had a medical history of PBC or fulfilled the diagnostic criteria of PBC at baseline, and 2076 (96.2%) individuals were non-PBC. After a median follow-up of 6.6 (interquartile range: 4.8–8.1) years, 0.6% (13 of 2076) developed PBC, with an accumulative 5-year incidence rate of 0.5%. LASSO regression showed that levels of alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), immunoglobulin M (IgM), eosinophilia proportion (EOS%), gamma globulin percentage, and hemoglobin (Hb) were predictors for developing PBC. Multivariate logistic regression analysis showed that in AMA-M2 positive individuals without PBC at baseline, five included difference indicators ALP (OR = 1.047, 95% CI: 1.034-1.061), GGT (OR = 1.014, 95% CI: 1.009-1.019), IgM (OR = 1.615, 95% CI: 1.170-2.291), ESO% (OR = 1.211, 95% CI: 1.056-1.379), and gamma globulin percentage (OR = 1.150, 95% CI: 1.022-1.284) were independent predictive risk factors for PBC in AMA-M2 positive physical examination population. Multivariate logistic regression is used to construct a predictive model based on 6 selected predictive factors from LASSO regression analyses, and ROC analysis showed that the area under the curve (AUC) of the model was 0.954 (95% CI: 0.933-0.976) in the training cohort, 0.975 (95% CI: 0.953-0.998) in the validation cohort 1, and 0.917 (95% CI: 0.847-0.987) in validation cohort 2.

Conclusion: The new onset of PBC in non-PBC AMA-M2 positive individuals at baseline was rare during six years of follow-up. The prediction model based on ALP, GGT, IgM, EOS%, gamma globulin percentage, and Hb has a certain predictive ability for the occurrence of PBC in this population, which can provide a reference for the early identification of high-risk individuals for PBC.

Part II: Safety and Enhanced Immunogenicity of COVID-19 Vaccine in Patients with Primary Biliary Cholangitis and Diabetes

Project Ⅴ. Research on the Safety and Immunogenicity of a Booster Dose of the Inactivated COVID-19 Vaccine in Patients with Primary Biliary Cholangitis

Objective: The coronavirus disease 2019 (COVID-19) vaccine is effective in controlling the epidemic and plays an important role in reducing the severe disease rate and mortality of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The inactivated vaccine is the most frequently used type of COVID-19 vaccine in China. Primary biliary cholangitis (PBC) is an autoimmune disease characterized by non-suppurative inflammation of small intrahepatic bile ducts and anti-mitochondrial antibodies. The occurrence of PBC can lead to the dysfunction of the immune system. The safety and immunogenicity of the inactivated SARS-CoV-2 vaccine in patients with PBC remains unclear. Therefore, this study aims to investigate the safety and immunogenicity of the inactivated SARS-CoV-2 vaccine in patients with PBC.

Methods: There were two cohorts of participants enrolled from the Peking Union Medical College Hospital in our study. One cohort includes 73 patients with PBC and 73 healthy controls (HCs) to investigate the antibody response to inactivated COVID-19 vaccine among patients with PBC. Another cohort is composed of 86 patients with PBC to conduct an online survey. The levels of anti-receptor-binding domain (RBD)-specific IgG, neutralizing antibody (NAb) toward SARS-CoV-2 wild type (WT), and NAb toward SARS-CoV-2 Omicron BA.4/5 subvariant were measured to evaluate the vaccine-induced immunological responses.

Results: The titers of anti-RBD-specific IgG (P = 0.013) and inhibition rates of NAb toward WT (P < 0.001) were significantly decreased in patients with PBC compared to HCs after booster vaccination for more than 6 months. Both HCs and patients with PBC showed poor resistance against BA.4/5 due to the detected inhibition rates being lower than the positive threshold. No laboratory parameters were significantly correlated with the three antibodies. Results from an online survey among PBC patients revealed that 24% of them received triple doses of the COVID-19 vaccine whilst 63% of them were unimmunized. The unvaccinated PBC patients were more worried about the safety of the COVID-19 vaccine compared with the vaccinated (P = 0.004). The main reason for patients with PBC not being vaccinated was “Doctors do not recommend vaccination”. The prevalence of adverse effects after the first dose of vaccine, the second dose of vaccine, and the third dose of vaccine were 6.1%, 10.3%, and 9.5% respectively. None of the patients with PBC reported life-threatening or serious adverse events requiring hospitalization after vaccination with the inactivated COVID-19 vaccine.

Conclusions: Patients with PBC showed impaired antibody responses after booster vaccination for more than 6 months. Decreased anti-BA.4/5 responses give rise to the possibility of breakthrough infections for both patients with PBC and HCs. The safety of the COVID-19 inactivated vaccine in patients with PBC is relatively safe, but it still needs to be verified in a larger cohort of PBC patients.

Project Ⅵ. Investigation into the Vaccination Status, Vaccination Attitudes and Safety of the COVID-19 Vaccine among Diabetic Patients in China

Objective: Diabetes mellitus (DM) is one of the most common chronic diseases in China. DM has a higher risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and is one of the risk factors for poor prognosis of coronavirus disease 2019 (COVID-19). The COVID-19 vaccine is one of the key measures to control the SARS-CoV-2 pandemic. However, the actual coverage of COVID-19 vaccination in DM patients in China and the related factors affecting vaccination behavior remain unclear. Therefore, this study aimed to investigate the actual coverage rate, vaccination attitude, and safety of the COVID-19 vaccine in Chinese patients with DM.

Methods: The questionnaire star platform was used to design an online questionnaire. From August 3, 2022, to October 3, 2022, a questionnaire survey based on cross-sectional study design was conducted among DM patients from 180 tertiary hospitals in China who were admitted to endocrinology departments to collect information about their vaccination status, adverse reactions after vaccination, and attitudes towards COVID-19 vaccine. Multiple logistic regression analysis model was used to explore the potential factors affecting COVID-19 vaccination behavior in patients with DM.

Results: A total of 2200 patients with DM were collected with valid questionnaires, and the results showed that 1,929 DM patients (87.7%) had received at least one dose of COVID-19 vaccine, and 271 DM patients (12.3%) had never received COVID-19 vaccine before participating in the survey. In addition, 65.2% of DM patients (n = 1434) had completed booster immunization with the COVID-19 vaccine, while 16.2% of DM patients (n = 357) had only finished fully-vaccination, and 6.3% of DM patients (n = 138) had only completed partial vaccination with COVID-19 vaccine. The adverse reaction rates of the first, second, and third doses of COVID-19 vaccine in DM patients were 6.0%, 6.0%, and 4.3%, respectively. Multiple logistic regression analysis showed that DM patients were complicated with immunoinflammatory diseases [partially vaccinated: Odds ratio (OR) = 0.12, 95% Confidence interval (CI): 0.02-0.99, P = 0.049; fully vaccinated: OR = 0.11, 95% CI: 0.02-0.51, P = 0.005; booster vaccinated: OR = 0.28, 95% CI: 0.12-0.64, P = 0.002], combined with diabetic nephropathy (partially vaccinated: OR = 0.23, 95% CI: 0.09-0.58, P = 0.048; fully vaccinated: OR = 0.51, 95% CI: 0.26-0.99, P = 0.048; booster vaccinated: OR = 0.30, 95% CI: 0.17-0.53, P < 0.001) and attitudes of COVID-19 vaccine safety (partially vaccinated: OR = 0.44, 95% CI: 0.22-0.87, P = 0.018; fully vaccinated: OR = 0.48, 95% CI: 0.28-0.82, P = 0.007; booster vaccinated: OR = 0.45, 95% CI: 0.29-0.70, P < 0.001) were the related factors affecting the three vaccination behavior of the COVID-19 vaccines.

Conclusion: This study shows that the actual coverage rate of the COVID-19 vaccine in patients with DM in China is high. The co-morbidities of patients with DM and the perception of COVID-19 vaccine safety influenced the COVID-19 vaccination behavior of patients with DM. In this study, no serious adverse events were observed in patients with DM after vaccination with the COVID-19 vaccine, indicating that COVID-19 vaccination is relatively safe for patients with DM.

Project Ⅶ. Research on the Immunogenicity of Type 2 Diabetes Patients Receiving Booster Inactivated COVID-19 Vaccine

Objective: Patients with type 2 diabetes mellitus (T2DM) are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and are at high risk of developing severe disease after infection. Inactivated vaccines are the most widely administered type of coronavirus disease 2019 (COVID-19) vaccine in China. However, the immunogenicity of T2DM patients after booster vaccination with inactivated COVID-19 vaccine remains unclear. This study aimed to investigate the antibody response of patients with T2DM to inactivated COVID-19 vaccine after enhanced immunization.

Methods: A total of 201 patients with T2DM and 102 healthy controls (HCs) who had been vaccinated with inactivated COVID-19 vaccine were included in this study. By detecting the anti-SARS-CoV-2 total antibodies, anti-receptor-binding domain (RBD)-specific IgG, neutralizing antibody (NAb) toward SARS-CoV-2 wild type (WT), NAb toward SARS-CoV-2 Omicron BA.4/5 subvariant to evaluate the humoral response induced by inactivated COVID-19 vaccine in patients with T2DM.

Results: There were no significant changes in anti-SARS-CoV-2 total antibodies, anti-RBD-specific IgG antibody, NAb toward SARS-CoV-2 WT and Omicron BA.4/5 levels between patients with T2DM and HCs within 6 months after booster immunization with inactivated COVID-19 vaccine. However, after 6 months of enhanced immunization with inactivated COVID-19 vaccine, the level of anti-RBD-specific IgG antibody (P = 0.018) and the inhibition rate of NAb toward SARS-CoV-2 WT (P = 0.007) in patients with T2DM were significantly lower than those in HCs. After booster immunization of patients with T2DM and HCs, the inhibition rate of NAb toward SARS-CoV-2 Omicron BA.4/5 subvariant was still lower than the minimum effective inhibition rate, resulting in the infection of patients with T2DM patients still unable to resist Omicron BA.4/5 strain. There was a significant positive correlation between the level of anti-RBD-specific IgG antibody and the proportion of CD3⁺CD4^-CD8^-T cells in the peripheral blood of patients with T2DM after booster immunization with inactivated COVID-19 vaccine (P = 0.045). In addition, the proportion of CD3⁺CD4^-CD8^-T cells in the peripheral blood of T2DM patients with positive anti-RBD-specific IgG antibody after booster immunization was significantly higher than that of T2DM patients with negative anti-RBD-specific IgG antibody (P = 0.005).

Conclusion: Within 6 months after booster immunization with inactivated COVID-19 vaccine, the response pattern of patients with T2DM to vaccine-induced antibodies is similar to that of HCs. However, the humoral response of patients with T2DM was impaired compared to HCs after more than 6 months of booster immunization with inactivated COVID-19 vaccine. In addition, patients with T2DM and HCs are still at risk of breakthrough infection with Omicron variants after booster immunization with inactivated COVID-19 vaccine.