Probing the binding specificities of human Siglecs by cell-based glycan arrays

Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.

Immune cells are equipped with an array of glycan-binding proteins (GPBs) capable of interpreting the biological information encoded by glycans. Endogenous GBPs recognize host-derived “self” and foreign-derived “nonself” glycans and produce cues that are integrated into the signaling network of immune cells and contribute to immune homeostasis and the immune response (1). Siglecs (sialic acid–binding immunoglobulin-like lectins) serve in self-recognition and transmit immune inhibitory signals upon binding to a select repertoire of sialoglycans expressed by host cells raising the threshold for immune activation (2, 3). The human Siglec family consists of 14 functionally expressed members, and these are composed of an N-terminal V-set immunoglobulin (Ig)-like domain that mediates sialoglycan binding followed by varying numbers of C2-set Ig-like domains. Intracellularly, most Siglecs have immunoreceptor tyrosine-based inhibition motifs, and Siglec-14/15/16 carry immunoreceptor tyrosine-based activation motifs (3–7). Siglecs are broadly expressed throughout the immune system, and several Siglecs are also found outside of the immune system, such as Siglec-4 (MAG), which is expressed by oligodendrocytes and Schwann cells in the nervous system (8). Although the diverse biological functions within and outside of the immune system of Siglecs are not fully understood, Siglecs generally contribute to immune homeostasis by dampening immune activation upon recognition of sialoglycans. For example, Siglec-2 (CD22) can suppress B cell receptor activation (9), and Siglec-9 can dampen neutrophil activation (10). Cancer cells with aberrant sialoglycans and pathogens that express sialic acids can exploit Siglec signaling to modulate immune responses (11, 12). Moreover, Siglec-3 (CD33) is strongly associated with risk for Alzheimer’s disease and expressed on microglia cells (13, 14). Given the potent immune modulatory functions of Siglecs and their wide involvement in autoimmunity, infection, cancer, and neurodegeneration, Siglecs are promising therapeutic targets (7, 15). However, many of the natural ligands of Siglecs have not been fully identified, and endogenous ligands for several Siglecs including Siglec-3/CD33 remain elusive.Human cells can produce a large diversity of glycans capped with sialic acids (Sia), a family of chemically diverse sugars with N-acetylneuraminic acid (Neu5Ac) being the predominant type in humans. Sialic acids are generally found at the termini of mammalian glycans, and most types of glycoconjugates including N-glycoproteins, multiple types of O-glycoproteins, and glycolipids carry oligosaccharides capped by sialic acids (16, 17). Sialylation is one of the most complex regulated steps in glycosylation with 20 distinct Golgi-located sialyltransferase isoenzymes dedicated to catalyze transfer of sialic acids to galactose (ST3GAL1-6, ST6GAL1 and 2), N-Acetylgalactosamine (Gal-NAc) (ST6GALNAC1-6), or sialic acid (ST8SIA1-6) via α2-3, α2-6, or α2-8 linkages, respectively and with different preferences for the underlying glycan structures and types of glycoconjugate (18–20). The resulting plethora of sialic acid-containing glycans constituting the sialome of cells provides a vast catalog of ligands for Siglecs and potential for distinct instructive cues for the immune response (16). The current insight into the interactome of Siglecs is largely derived from studies with libraries of synthetic and natural glycans printed on glass arrays (21, 22). These glycan arrays have demonstrated distinct structural glycan features that drive selective binding of individual Siglecs, including the linkage type of sialic acids, the core disaccharide carrying sialic acids, and glycan modifications such as sulfation or acetylation (23–27). However, printed glycan arrays may not present glycans in the natural context of the overall glycoconjugate structure and the cell surface with spatial organization and competition dynamics limiting insight into the fine binding specificities of Siglecs and their interactions with the host cell sialome.Here, we took advantage of our recently developed cell-based glycan array strategy (28–30) and generated an expanded sialome sublibrary with the human embryonic kidney (HEK) 293 for dissection of Siglec binding properties. First, combinatorial gene knockout (KO) was used to delete distinct subsets of sialyltransferase isoenzymes or all endogenous sialylation capacity. Second, using targeted gene knock-in (KI), individual sialyltransferase isoenzymes were introduced in the absence of other isoenzymes. Finally, we introduced selected sulfotransferase isoenzymes to explore cross-talk between sialylation and sulfation. To specifically address the influence of clustered O-glycan presentation for Siglec binding, we introduced a large panel of reporter constructs designed to display human O-glycodomains derived from mucins and mucin-like O-glycoproteins with different densities and patterns of O-glycans. The cell-based sialome array reproduced previous results for binding specificities for Siglec-2 (CD22) and Siglec-9 and led to insight into the binding specificities of Siglec-4/7/15 for distinct GalNAc-type O-glycans and their presentation on O-mucin–like glycoproteins. Finally, we demonstrate that Siglec-3/7/8/15 have preferential binding to sulfated sialoglycans yet have different specificities for underlying glycoconjugate structures. We further discovered the 6′-Su-SLacNAc (Neu5Acα2-3[6-O-sulfo]Galβ1-4GlcNAc) epitope on N-glycans and glycolipids as the ligand for Siglec-3/CD33 as well as Siglec-8. In summary, the cell-based display of the human sialome enables dissection of the Siglec interactome in the natural context of a human cell and provides the biosynthetic and genetic basis for the identified ligands.     

肩关节稳定机制再平衡理论在肩关节不稳和运动功能障碍治疗中的临床应用

目的介绍肩关节稳定机制的新理论——再平衡理论，以及该理论在临床中的应用。方法通过广泛查阅近年来肩关节不稳和功能障碍的临床研究文献，结合本课题组临床实践经验，总结肩关节静力和动力稳定机制的相互作用。结果通过回顾文献并总结临床经验，笔者提出了“肩关节稳定机制再平衡理论”，即当肩关节稳定机制破坏后，肩关节稳定可以通过肩关节静力稳定机制和动力稳定机制的再平衡来实现。在肩关节动力稳定失去平衡时，可通过动力稳定机制不同组织之间再平衡和/或静力稳定机制组织加强，恢复肩关节动力性稳定；在肩关节静力稳定失去平衡时，通过肩关节静力稳定机制中软组织和骨结构的再平衡来恢复肩关节静力性稳定。结论肩关节稳定机制再平衡理论是用整体、运动的观点来认识肩关节不稳和运动功能障碍的发生、发展和治疗后的转归，可以有效指导临床上肩关节不稳或运动功能障碍等涉及肩关节稳定机制疾患的治疗。     

黄芩苷调节IL-33/ST2信号通路对糖尿病视网膜病变大鼠视网膜新生血管生成的影响

目的 探讨黄芩苷调节白细胞介素(IL)-33/基质裂解素2(ST2)信号通路对糖尿病视网膜病变(DR)大鼠视网膜新生血管生成的影响。方法 SD大鼠随机分为对照组(正常大鼠,灌胃生理盐水)、DR模型组(大鼠建立DR模型后,灌胃生理盐水)和黄芩苷低、中、高剂量组(大鼠建立DR模型后,分别灌胃75 mg·kg^-1、150 mg·kg^-1、300 mg·kg^-1黄芩苷),所有大鼠均以右眼为实验眼。FFA检查各组大鼠视网膜血管生成情况,ELISA检测各组大鼠血清血管内皮生长因子(VEGF)、Ang-1、IL-6、IL-33、肿瘤坏死因子-α(TNF-α)水平,Western blot检测各组大鼠视网膜组织中IL-33、ST2蛋白表达水平。结果 与对照组相比,DR模型组大鼠血清VEGF、Ang-1、IL-6、IL-33、TNF-α水平及视网膜组织中IL-33、ST2蛋白表达水平均显著升高(均为P<0.05),右眼眼底新生血管增多,荧光素渗漏明显;与DR模型组相比,黄芩苷低、中、高剂量组大鼠血清VEGF、Ang-1、IL-6、I...     

Right Ventricular Function Evolution With Pregnancy in Repaired Tetralogy of Fallot

This case illustrates the evolution of right ventricular (RV) 3-dimensional (3D) area strain during pregnancy in a patient with repaired Tetralogy of Fallot. The report highlights impairment in RV function with pregnancy, suggesting the importance of prepregnancy RV systolic function assessment, especially using 3D echocardiography.     

牙周基础治疗联合抗生素对中重度慢性牙周炎患者IL-33、IL-6的影响研究

目的探讨牙周基础治疗联合抗生素对中重度慢性牙周炎患者IL-33、IL-6的影响。方法选取上海市闵行区浦江镇社区卫生服务中心2016年12月—2018年12月接诊的78例中重度慢性牙周炎患者进行研究。根据治疗方法的不同,将其均分为对照组和观察组各39例。对照组采取牙周基础治疗,观察组在对照组的基础上联合抗生素治疗。比较分析两组患者治疗后的有效率,治疗前和治疗后3个月的白细胞介素-33(IL-33)、白细胞介素-6(IL-6)、C-反应蛋白(CRP)、肿瘤坏死因子(TNF-α)水平、牙周指标(牙龈指数、龈沟出血指数、探诊后出血指数以及牙周袋深度)以及不良反应情况。结果观察组患者治疗总有效率(94.87%)明显高于对照组(79.48%),差异有统计学意义(P0.05)。治疗前,两组患者的IL-33、IL-6、CRP、TNF-α、GI、SBI、BOP和PD水平差异无统计学意义(P0.05)。治疗3个月后,观察组IL-33、IL-6、CRP、TNF-α、GI、SBI、BOP和PD水平均明显低于对照组,差异有统计学意义(P0.05)。观察组不良反应发生率明显低于对照组,差异有统计学意义(P0.05)。结论牙周基础治疗联合抗生素能够改善中重度慢性牙周炎患者的血清炎性因子水平和牙周指标,且预后良好,值得临床上推广应用。     

股骨转子间骨折的稳定性重建概念演化与研究进展

目的总结近年来股骨转子间骨折在稳定性重建方面的概念演化与研究进展。方法查阅国内外相关文献并结合自身经验，从股骨转子间骨折的解剖特点、稳定型骨折与不稳定型骨折分类、稳定性复位与不稳定性复位、术中加压初始稳定与术后滑动二次稳定、内固定术后稳定性评估、早期下地站立负重等方面进行总结分析。结果股骨转子间骨折发生于股骨颈干骺端转换区，具有天然的内翻不稳定倾向。骨折复位质量是影响后续内固定物安放的最重要前提因素。判断骨折复位质量有对线和对位两方面，对线采用 Garden 指数；在对位方面，随着皮质对位理念（正性、中性、负性）的提出，特别强调前内侧皮质的相互砥住支撑（解剖、正性），是获得骨折稳定性复位的关键，而不再强调后内侧小转子骨块的作用。术后影像学的稳定性评分为早期下地站立负重提供了量化指标。但术中的前内侧皮质支撑复位，在术后头颈骨块滑动获得二次稳定的过程中，仍有皮质对位丢失现象，需研究其危险因素和防范措施。结论股骨转子间骨折在取得良好对线的基础上，只要获得了前内侧皮质的相互砥住和支撑，并用内固定器械维持住，就获得了术后稳定性。术后稳定性评分优良者，可以安全地早期下地负重、站立行走活动。     

Transient retrograde interfragmentary compression technique in AO/OTA type 33-C distal femur fractures: A surgical technique and short-term radiographic follow up results

Surgical treatment of AO/OTA type 33-C fractures is a therapeutic challenge despite advances in surgical instruments and techniques. We introduce a novel surgical technique named transient retrograde interfragmentary compression (TRIC) to help intraarticular fragment reduction in AO/OTA type 33-C fracture. We inserted a partial threaded 7.0-cannulated screw with a washer along the transepicondylar axis from the medial femoral epicondyle during the articular block reduction process of AO/OTA type 33-C fractures to strengthen the compressive force between the condylar fragments and to enhance the handling of the articular block fragment in the alignmental correction stage. Following the provisional reduction and fixation using lateral distal femur locking compression plate, TRIC screw was removed. Fifteen AO/OTA type 33-C distal femoral intraarticular fractures of thirteen patients were surgically treated using the TRIC technique. We analyzed the radiographic result of the patients by measuring the horizontal gap and vertical step-off in the postoperative radiographs. Mean horizontal fracture gap was 0.34 mm and mean vertical step-off between bicondylar fragments was 0.63 mm. The median value of the horizontal fracture gap and vertical step off was 0 and 0.46 mm, respectively. Mean time to union in the bicondylar fracture fragment was 9 week. TRIC is considered to be a valuable surgical reduction technique in the treatment of the AO/OTA 33-C type fractures.