Early signatures of bleeding and mortality in patients on left ventricular assist device support: novel methods for personalized risk-stratification

Background: Left ventricular assist devices (LVADs) provide support for patients with end-stage heart failure. The aims of this study were to determine whether baseline analysis and early trends in routine laboratory data, platelet activity, and thromboinflammatory biomarkers following LVAD implantation reveal trends that predict personalized risks of one-year gastrointestinal (GI) bleeding, stroke, pump thrombosis, drive-line infections and mortality in patients on LVAD support.

Methods: We performed an observational study at the University of Kentucky with 61 participants who underwent first-time LVAD implantation. Blood was collected at baseline and post-op days 0, 1, 3 and 6 as well as clinical follow-up. Demographics, clinical characteristics, one-year adverse events and routine laboratory data were collected from electronic medical records. Platelet function and plasma biomarkers were profiled.

Results: Evaluation of routine laboratory results revealed that sustained thrombocytopenia and increased mean platelet volume (MPV) were associated with development of GI bleeding and mortality. Platelet function at follow-up visit predicted one-year bleeding events. Thrombotic biomarker sCD40L strongly predicted one-year GI bleeding at baseline before implantation and within the first week following LVAD implant.

Conclusions: Early trends in routine bloodwork and platelet function may serve as novel signatures of patients at risk to experience adverse events.     

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome(SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11(SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum(ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1(LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall,these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.     

阿加曲班用于肝素相关性血小板减少症患者PCI 术抗凝治疗1 例 并文献复习

目的:探讨肝素相关性血小板减少症(heparin-induced thrombocytopenia,HIT)患者行经皮冠状动脉介入治疗(percutaneoustransluminal coronary intervention,PCI)术中及术后使用阿加曲班进行抗凝的效果及安全性。方法:报道并回顾性分析阿加曲班用于肝素相关性血小板减少症患者PCI术一例并进行文献复习。结果:患者女性,55岁,皮下注射低分子肝素4天后血小板由140×109/L下降为17×109/L,患者出现牙龈出血、左前臂出现瘀斑,诊断为HIT。行冠状动脉支架植入术术中及术后使用阿加曲班抗凝,未出现出血及血栓事件。结论:肝素相关性血小板减少症患者行PCI术,术中及术后采用阿加曲班进行抗凝治疗是有效安全的。     

利福平导致严重血小板减少1例

利福平主要用于结核病的治疗,能引起血小板减少等不良反应。本病例使用利福平后出现严重血小板减少,血小板下降至4×10⁹/L,立即停用利福平并输注血小板后血小板恢复正常。因此,在利福平使用过程中应密切观察病情,监测血常规、肝肾功能等,及时发现不良反应,必要时立即停药,并对血小板明显下降者(<30×10⁹/L)给予补充血小板等治疗。对明确由利福平引起血小板减少者,治疗时应不再使用该药,以避免药物不良事件的发生。     

Entry of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a globe-shaped virus covered by a dense icosahedral array of glycoproteins Gn/Gc that mediate the attachment of the virus to host cells and the fusion of viral and cellular membranes. Several membrane factors are involved in virus entry, including C-type lectins and nonmuscle myosin heavy chain ⅡA. The post-fusion crystal structure of the Gc protein suggests that it is a class Ⅱ membrane fusion protein, similar to the E/E1 protein of flaviviruses and alphaviruses. The virus particles are internalized into host cell endosomes through the clathrin-dependent pathway, where the low pH activates the fusion of the virus with the cell membrane. With information from studies on other bunyaviruses, herein we will review our knowledge of the entry process of SFTSV.     

Renal Impairment in Hepatic Cryotherapy

Cryoshock is a syndrome of coagulopathy, renal, and pulmonary injury following cryotherapy, and its etiology is unknown. The aim of this study was to assess the impact of hepatic cryotherapy on renal function, and whether this effect is related to volume of cryotherapy, and to identify any predictors of renal impairment in patients who undergo cryotherapy. A retrospective analysis of all patients with primary or secondary hepatic malignancy treated with cryotherapy from April 1990 to October 1996 was conducted. Ten of 204 patients with renal impairment (elevation in creatinine of greater than 0.02 mmol/L for more than 2 days postprocedure) were identified. One patient had postoperative pancreatitis with late renal impairment (20 days) and was excluded. The severity of renal impairment was usually modest (mean rise in creatinine of 0.31 mmol/L; SD, 0.19). Two patients required temporary hemodialysis. Only one patient, who had significant cardiac disease, had associated pulmonary injury and shock. Demographic data in both groups were comparable, except for a trend toward more noncolorectal cancer patients in the renal impairment group (4/9 vs 33/194). Patients in the renal impairment group had a greater number of lesions than those of the nonrenal impairment group (3.4 vs 2.1,P< 0.01), as well as larger lesion diameter (2.9 vs 1.9,P< 0.01), increased freezing time (74.7 vs 44.3,P< 0.01), and a higher aspartate transaminase (AST) (2254 vs 1157,P< 0.01). This study suggests that renal impairment is more likely to be seen in patients undergoing more extensive cryotherapy. The number and diameter of lesions together with AST data link renal injury with magnitude of liver injury—all renal impairment patients had an AST > 1000, compared with only 28% of patients who did not.     

Gene therapy for PIDs: Progress,pitfalls and prospects

Substantial progress has been made in the past decade in treating several primary immunodeficiency disorders (PIDs) with gene therapy. Current approaches are based on ex-vivo transfer of therapeutic transgene via viral vectors to patient-derived autologous hematopoietic stem cells (HSCs) followed by transplantation back to the patient with or without conditioning. The overall outcome from all the clinical trials targeting different PIDs has been extremely encouraging but not without caveats. Malignant outcomes from insertional mutagenesis have featured prominently in the adverse events associated with these trials and have warranted intense pre-clinical investigation into defining the tendencies of different viral vectors for genomic integration. Coupled with issues pertaining to transgene expression, the therapeutic landscape has undergone a paradigm shift in determining safety, stability and efficacy of gene therapy approaches. In this review, we aim to summarize the progress made in the gene therapy trials targeting ADA-SCID, SCID-X1, CGD and WAS, review the pitfalls, and outline the recent advancements which are expected to further enhance favourable risk benefit ratios for gene therapeutic approaches in the future.     

Conformational stability and domain unfolding of the Von Willebrand factor A domains

Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.     

Crystallographic structure of the human leukocyte antigen DRA, DRB3*0101: models of a directional alloimmune response and autoimmunity

We describe structural studies of the human leukocyte antigen DR52a, HLA-DRA/DRB3*0101, in complex with an N-terminal human platelet integrin alphaII(B)betaIII glycoprotein peptide which contains a Leu/Pro dimorphism. The 33:Leu dimorphism is the epitope for the T cell directed response in neonatal alloimmune thrombocytopenia and post-transfusion purpura in individuals with the alphaII(B)betaIII 33:Pro allele, and defines the unidirectional alloimmune response. This condition is always associated with DR52a. The crystallographic structure has been refined to 2.25 A. There are two alphabeta heterodimers to the asymmetric unit in space group P4(1)2(1)2. The molecule is characterized by two prominent hydrophobic pockets at either end of the peptide binding cleft and a deep, narrower and highly charged P4 opening underneath the beta 1 chain. Further, the peptide in the second molecule displays a sharp upward turn after pocket P9. The structure reveals the role of pockets and the distinctive basic P4 pocket, shared by DR52a and DR3, in selecting their respective binding peptide repertoire. We observe an interesting switch in a residue from the canonically assigned pocket 6 seen in prior class II structures to pocket 4. This occludes the P6 pocket helping to explain the distinctive "1-4-9" peptide binding motif. A beta57 Asp-->Val substitution abrogates the salt-bridge to alpha76 Arg and along with a hydrophobic beta37 is important in shaping the P9 pocket. DRB3*0101 and DRB1*0301 belong to an ancestral haplotype and are associated with many autoimmune diseases linked to antigen presentation, but whereas DR3 is susceptible to type 1 diabetes DR52a is not. This dichotomy is explored for clues to the disease.     

儿童免疫性血小板减少症血清中抗核仁抗体纯化以及靶抗原的筛选

免疫性血小板减少症为儿童常见获得性免疫性疾病,本研究前期发现有较高比例的抗核仁抗体阳性的儿童为免疫性血小板减少症患者。为筛选鉴定抗核仁抗体阳性免疫性血小板减少症患儿血清中抗核仁抗体识别的靶抗原,本研究利用HEP-2细胞系建立了细胞固定-抗体结合-抗体洗脱-抗体中和体系,成功纯化免疫性血小板减少症患儿血清中抗核仁抗体。利用蛋白免疫共沉淀和质谱分析法筛选出Ncl、Krt1、Krt2、Krt10等蛋白可能为免疫性血小板减少症患儿血清中抗核仁抗体结合的靶抗原。这些结果为免疫性血小板减少症患儿血清中抗核仁抗体靶抗原的鉴定提供有效方法,可进一步深化我们对免疫性血小板减少症发病机制的认识。