胎盘Hofbauer细胞在HBV母婴垂直传播的作用

目的:探讨胎盘Hofbauer细胞在乙型肝炎病毒(HBV)母婴垂直传播的作用。方法:垂直传播组(母亲及其新生儿血HBsAg和HBV-DNA均为阳性)、非垂直传播组(母亲血HBsAg和HBV-DNA阳性而新生儿阴性)和对照组(母亲及其新生儿血HBsAg和HBV-DNA均为阴性)各30例,透射电了显微镜观察其胎盘Hofbauer细胞超微结构变化及其与HBV颗粒的关系。结果:①对照组未发现Hofbauer细胞:在垂直传播组和非垂直传播组,Hofbauer细胞散在分布于胎盘间质中。②在垂直感染组,Hofba-uer细胞肿胀,胞浆突起减少,胞浆空泡变大;粗面内质网及高尔基复合体萎缩,线粒体缩小,溶酶体少见;胞核增大,染色质浓缩、边聚。在非垂直感染组,Hofbauer细胞呈圆形或卵圆形,细胞表面有大量胞浆突起,排列着微吞饮小体,细胞胞浆内有圆形空泡;线粒体呈杆状,峭排列紧密,溶酶体较多,高尔基体及粗面内质网欠发达;胞核偏位,核仁显著,染色质分布均匀。③在Hofbauer细胞胞质空泡、细胞间隙内存在单个或多个成熟病毒颗粒、病毒包涵体和病毒抗原颗粒。结论:HBV可引起胎盘Hofbauer细胞超微结构改变,并经Hofbauer细胞介导引起母婴垂直传播。     

构建表达Flag-GPI的重组乙型肝炎病毒载体

乙型肝炎病毒(hepatitis B virus,HBV)是小型的嗜肝DNA病毒,能够特异性地感染人肝实质细胞。HBV的易感性可以归于多个方面,包括细胞表面的受体以及细胞内的蛋白或其他因子,目前对HBV易感性的了解还有待深入。本课题利用课题组原有的HBV 5c3c载体和糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)的特性,构建了重组HBV载体5c3c-CD59-Flag-GPI和5c3cT-Flag-GPI,转染Huh7细胞后可以将Flag标签锚定在细胞膜上,且可利用Flag抗体通过流式细胞术对其进行分选。在回补HBV包膜蛋白的情况下,5c3cT-Flag-GPI能包装形成完整的重组HBV颗粒。本研究为后续优化重组HBV的包装效率,进而为HBV易感性研究提供工具并奠定基础。     

趋化因子在HBV感染发病中的作用

乙型肝炎是一种以局部炎性为主的感染性疾病,乙型肝炎病毒（HBV）感染宿主细胞后可诱导宿主细胞中趋化因子分泌及其受体表达,趋化因子／受体的相互作用进一步介导中性粒细胞、淋巴细胞等向炎症部位聚集,参与组织损伤;同时诱导T、B细胞分化成熟,对乙型肝炎的发展与转归、肝组织的损伤与修复有重要影响。HBV引发的慢性乙型肝炎（CHB）以Th1细胞性炎性反应为主,研究表明乙型肝炎中某些趋化因子在肝脏高表达,其受体CXCR3和CCR5在Th1细胞高表达。趋化因子尤其是CXC和CC亚家族趋化因子在趋化Th1细胞中发挥重要的作用：     

整合素与配体结合的特征决定细胞游走速度

整合素与配体结合的特征决定细胞游走速度高等动物细胞在组织中的游走主要由细胞表面的粘附受体如整合素（ｉｎｔｅｇｒｉｎｓ）介导。整合素的作用包括：将细胞与胞外基质配体连结,传递作用力和细胞运动的必须信号。细胞在特定基质中是否能游走及游走速度取决于整合素与...     

乙型肝炎病毒受体研究进展

乙型肝炎(乙肝)病毒(hepatitis B virus,HBV)是嗜肝DNA病毒科的原型病毒.目前,对HBV的基因组成、复制过程、抗原结构及生物学特性等已有相当程度的了解[1].但由于缺乏合适的感染系统,对HBV感染的早期过程,如病毒如何结合靶细胞上的受体最终进入细胞等,仍不很清楚.     

HBsAb标记胶体金探针对HBV阳性血清感染HPT-8细胞的示踪研究

目的:为临床诊断提供HBV进入HPT-8的依据。方法:HBV阳性血清(1.5×10~8拷贝/毫升)体外感染HPT-8细胞48h,设感染组、阴性对照组和空白对照组,ELISA检测细胞培养上清中HBsAg、HBeAg,提取感染后传代的各代细胞中DNA用于PCR检测HBV DNA。免疫荧光定量PCR检测细胞培养上清中HBV DNA的滴度。采用乙肝表面抗原单克隆抗体标记的胶体金探针对感染的HPT-8细胞中的乙肝表面抗原进行标记示踪。结果:ELISA检测感染组细胞第1、2代培养上清中HBsAg阳性,PCR检测感染组第1、2代细胞HBV DNA,结果均阳性。免疫荧光定量PCR检测感染组细胞上清在第1、2代、3代48h的HBV滴度分别为8×10~4、4.6×10~3、<10~3(拷贝/毫升)。通过胶体金探针的标记:乙肝表面抗原存在于感染细胞的溶酶体内、细胞膜和绒毛上。结论:血清中的HBV病毒能进入胎盘滋养细胞,并可从感染滋养细胞的树脂切片中获得乙肝表面抗原的超微结构定位。     

免疫检查点抑制剂在慢性乙型肝炎中的研究进展

慢性乙型肝炎病毒(hepatitis B virus,HBV)感染仍是全球主要公共卫生问题之一。尽管目前已有预防性疫苗可有效预防新发HBV感染,但全球仍有约2.5亿慢性HBV感染者,其中每年约有100多万人死于HBV相关的慢性肝病,形势仍不容乐观。抗病毒药物(干扰素和核苷类似物等)可抑制病毒复制,降低乙肝相关并发症,但由于其存在耐药性难以达到临床终点。免疫检查点抑制剂作为逆转T细胞耗竭的重要策略,重建有效的功能T细胞反应将是治疗慢性乙肝患者一种有前景的免疫调节方法。本文总结了程序性死亡受体1/细胞程序性死亡-配体1(PD-1/PD-L1)、细胞毒性T淋巴细胞相关蛋白4(CTLA-4)、T细胞免疫球蛋白和ITIM结构域(TIGIT)、T细胞免疫球蛋白和粘蛋白域蛋白-3 (Tim-3)、淋巴细胞活化基因-3(lag-3)五种免疫检查点分子的抑制剂在慢性乙型肝炎中的重要研究进展。     

乙型肝炎病毒受体研究进展

肝细胞是乙型肝炎病毒(HBV)感染的主要靶器官,病毒黏着并进一步侵入肝细胞是感染启动的关键步骤。近几年的研究发现,肝细胞膜上的受体在病毒感染中起着至关重要的作用。我们对近年来几种可能的HBV受体的研究情况进行综述,对阐明HBV入侵肝细胞的机制具有一定的意义。     

免疫检查点阻断在逆转慢性结核病T细胞耗竭中的应用

抗原特异性T细胞是机体抗感染和抗肿瘤免疫应答的关键因素。持续的抗原刺激可能导致T细胞耗竭,其特征为效应性T细胞应答能力变弱、细胞因子分泌能力降低、抑制性受体持续表达和转录状态改变等。现有研究表明,免疫检查点阻断可逆转肿瘤和HIV、HBV等慢性感染过程中的T细胞耗竭,但是其在逆转慢性结核病T细胞耗竭中的作用尚不明确。该综述讨论了持续性结核菌感染诱导T细胞耗竭的发生机制,慢性结核菌感染中表达上调的抑制性受体CTLA-4、PD-1、TIM-3、LAG-3、KLRG1、2B4和BTLA的研究现状,预防和扭转T细胞耗竭状态以增强结核菌控制的策略等。这些研究将对靶向T细胞耗竭的结核病免疫治疗有重要提示。     

乙型肝炎病毒进入肝细胞机制研究进展

乙型肝炎病毒(hepatitis B virus,HBV)感染早期进入肝细胞机制研究一直是HBV研究领域的热点和难点．简单易得的HBV体外感染细胞模型是HBV感染进入机制研究无法逾越的主要障碍．近年来,随着新型HBV体外感染细胞模型的建立和应用(HepRG细胞和树鼩原代肝细胞),HBV的进入机制研究取得了一系列重大发现．综述了近几年HBV进入肝细胞机制的最新研究进展,主要包括HBV表面蛋白进入相关结构域的鉴定,已发现的候选HBV进入相关分子和尚待解决的问题．     

A positive feedback synapse from retinal horizontal cells to cone photoreceptors

Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca(2+) and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca(2+), whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement.     

Cell membrane permeabilization via connexin hemichannels in living and dying cells

Vertebrate cells that express connexins likely express connexin hemichannels (Cx HCs) at their surface. In diverse cell types, surface Cx HCs can open to serve as a diffusional exchange pathway for ions and small molecules across the cell membrane. Most cells, if not all, also express pannexins that form hemichannels and increase the cell membrane permeability but are not addressed in this review. To date, most characterizations of Cx HCs have utilized cultured cells under resting conditions have and revealed low open probability and unitary conductance close to double that of the corresponding gap junction channels. In addition, the cell membrane permeability through Cx HCs can be markedly affected within seconds to minutes by various changes in the intra and/or extracellular microenvironment (i.e., pH, pCa, redox state, transmembrane voltage and intracellular regulatory proteins) that affect levels, open probability and/or (single channel) permeability of Cx HC. Net increase or decrease in membrane permeability could result from the simultaneous interaction of different mechanisms that affect hemichannels. The permeability of Cx HCs is controlled by complex signaling cascades showing connexin, cell and cell stage dependency. Changes in membrane permeability via hemichannels can have positive consequences in some cells (mainly in healthy cells), whereas in others (mainly in cells affected by acquired and/or genetic diseases) hemichannel activation can be detrimental.     

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.     

Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.     

No evidence of UV cone input to mono- and biphasic horizontal cells in the goldfish retina

Many animal species make use of ultraviolet (UV) light in a number of behaviors, such as feeding and mating. The goldfish (Carassius auratus) is among those with a UV photoreceptor and pronounced UV sensitivity. Little is known, however, about the retinal processing of this input. We addressed this issue by recording intracellularly from second-order neurons in the adult goldfish retina. In order to test whether cone-driven horizontal cells (HCs) receive UV cone inputs, we performed chromatic adaptation experiments with mono- and biphasic HCs. We found no functional evidence of a projection from the UV-sensitive cones to these neurons in adult animals. This suggests that goldfish UV receptors may contact preferentially triphasic HCs, which is at odds with the hypothesis that all cones contact all cone-driven HC types. However, we did find evidence of direct M-cone input to monophasic HCs, favoring the idea that cone–HC contacts are more promiscuous than originally proposed. Together, our results suggest that either UV cones have a more restricted set of post-synaptic partners than the other three cone types, or that the UV input to mono- and biphasic HCs is not very pronounced in adult animals.     

A glycosylated type I membrane protein becomes cytosolic when peptide: N-glycanase is compromised

The human cytomegalovirus-encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N-glycanase-type activity. siRNA molecules designed to inhibit the expression of the light chain, beta(2)-microglobulin, block the dislocation of Class I MHC molecules, which implies that US2-dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N-glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2-expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA-treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N-linked type I membrane glycoprotein in the cytosol. N-glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.