G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

The host defense response critically depends on the production of leukocytes by the marrow and the controlled delivery of these cells to relevant sites of inflammation/infection. The cytokine granulocyte-colony stimulating factor (G-CSF) is commonly used therapeutically to augment neutrophil recovery following chemo/radiation therapy for malignancy, thereby decreasing infection risk. Although best known as a potent inducer of myelopoiesis, we previously reported that G-CSF also promotes the delivery of leukocytes to sites of inflammation by stimulating expression of potent E-selectin ligands, including an uncharacterized ∼65-kDa glycoprotein. To identify this ligand, we performed integrated biochemical analysis and mass spectrometry studies of G-CSF–treated primary human myeloid cells. Our studies show that this novel E-selectin ligand is a glycoform of the heavy chain component of the enzyme myeloperoxidase (MPO), a well-known lysosomal peroxidase. This specialized MPO glycovariant, referred to as “MPO–E-selectin ligand” (MPO–EL), is expressed on circulating G-CSF–mobilized leukocytes and is naturally expressed on blood myeloid cells in patients with febrile leukocytosis. In vitro biochemical studies show that G-CSF programs MPO–EL expression on human blood leukocytes and marrow myeloid cells via induction of N-linked sialofucosylations on MPO, with concomitant cell surface display of the molecule. MPO–EL is catalytically active and mediates angiotoxicity on human endothelial cells that express E-selectin. These findings thus define a G-CSF effect on MPO chemical biology that endows unsuspected functional versatility upon this enzyme, unveiling new perspectives on the biology of G-CSF and MPO, and on the role of E-selectin receptor/ligand interactions in leukocyte migration and vascular pathology.Granulocyte-colony stimulating factor (G-CSF) serves a critical role in native immunity by directing elaboration of myeloid cells and their cytocidal arsenal (1), and the characteristic leukocytosis (the “leukemoid reaction”) of inflammatory reactions is fueled by physiologic up-regulation of G-CSF (2). Binding of G-CSF to its receptor on early hematopoietic progenitors and on intermediate-stage myeloid progenitors activates proliferation pathways, resulting in programmed expansion and release (“mobilization”) of progenitors and mature myeloid cells into the peripheral circulation (2). This cytokine is in routine clinical use, administered to stimulate myelopoiesis after chemo- or radiotherapy, to treat cyclic neutropenia, and to mobilize progenitor cells for hematopoietic stem cell transplantation (HSCT) (3).Leukocyte production stimulated by G-CSF is not sufficient to mediate host defense, as it is essential to deliver the leukocyte cytocidal arsenal at pertinent sites of inflammation. Leukocyte recruitment begins with tethering and rolling of blood-borne cells on the endothelial lining under hemodynamic shear conditions, followed by activation of integrins and firm adhesion to the vessel wall, culminating in transendothelial migration (4). The initial shear-resistant adherence of leukocytes to the endothelial surface is principally mediated by selectin receptor/ligand interactions (4). The selectin family consists of “leukocyte-specific” L-selectin and “vascular selectins” P- and E-selectin, each of which binds to specialized carbohydrate determinants containing an α2,3-linked sialic acid substitution on galactose, and an α1,3-linked fucose modification on N-acetylglucosamine, prototypically displayed as sialyl Lewis x (sLex), a tetrasaccharide recognized by monoclonal antibody, clone HECA-452 (4). Various glycoproteins that display sLex have been described as E-selectin ligands in mouse and human leukocytes. The principal E-selectin ligand on human and mouse myeloid cells is the molecule known as cutaneous lymphocyte antigen (CLA), a heavily sialofucosylated glycoform of P-selectin glycoprotein ligand-1 (PSGL-1) (5, 6). A molecule known as E-selectin ligand-1 (ESL-1) serves as an E-selectin ligand on mouse neutrophils (7), but it is not expressed on human neutrophils nor on human hematopoietic stem and progenitor cells (HSPCs) (8). Biochemical assays have indicated that the leukocyte β₂ integrins LFA-1 (lymphocyte function-associated antigen 1 or CD11a/CD18) and MAC-1 (macrophage-1 antigen or CD11b/CD18) can serve as E-selectin ligands on human leukocytes (9). CD43 has also been implicated to be an E-selectin ligand on mouse neutrophils and activated T cells (10), and we have reported that CD43 on human T cells can bind E-selectin, but not P-selectin (11). On human hematopoietic cells, two glycoproteins function as major counter receptors for E-selectin: the CLA molecule (5, 6) and a glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL) (6, 12). Studies from our laboratory have shown that following clinical G-CSF administration to mobilize hematopoietic progenitors for HSCT, circulating myeloid cells exhibit increased adherence to TNF-stimulated vascular endothelium compared with native leukocytes (NLs). Biochemical analysis showed that this augmented endothelial adherence of G-CSF–mobilized human leukocytes (MLs) was due to increased E-selectin ligand activity, in part mediated by expression of a novel E-selectin ligand, a membrane glycoprotein with a molecular weight of ∼65 kDa (12).To identify this previously uncharacterized E-selectin ligand, we used lectin affinity chromatography, combined with mass spectrometry peptide fingerprinting and Western blot analysis. Our studies show that this G-CSF–induced glycoprotein is a unique glycoform of myeloperoxidase (MPO), designated here as “MPO–E-selectin ligand” (MPO–EL), which contains E-selectin binding determinants on the ∼65-kDa heavy chain. Although MPO is usually expressed within the lysosome (azurophilic granules), our data show that G-CSF induces cell membrane expression of MPO concomitant with the elaboration of a glycovariant of the heavy chain, carrying specialized N-linked sialofucosylated determinants. MPO–EL is catalytically active and coincubation of MPO–EL-bearing myeloid cells with E-selectin–bearing endothelial cells engenders angiotoxicity. Our findings thus highlight the capacity of posttranslational carbohydrate modifications to shape protein function, revealing previously unrecognized versatility of MPO biology tuned by G-CSF that could incite vascular injury in vivo.     

反向脉冲谐波实时超声造影诊断背驼式肝移植术后肝静脉及下腔静脉并发症

目的探讨实时超声造影检查在背驼式肝移植术后肝静脉及下腔静脉并发症诊断中的应用价值。方法对25例背驼式肝移植术后CDFI疑诊肝静脉及下腔静脉并发症的患者进行实时超声造影检查。采用造影剂声诺维（SonoVue）2.4ml肘部浅静脉团注,反向脉冲谐波低机械指数（MI0.15-0.19）实时超声造影。将超声造影结果与增强CT对照分析。结果超声造影诊断下腔静脉血栓3例;肝静脉血栓5例,其中2例继发肝组织淤血、坏死;超声造影诊断肝静脉狭窄6例,下腔静脉狭窄4例,其中最严重的1例肝上下腔静脉内径3.8mm。CDFI与增强CT的符合率为72%（18/25）,实时超声造影与增强CT的符合率为100%（18/18）。结论实时超声造影对背驼式肝移植术后肝静脉及下腔静脉并发症的诊断能力与增强CT相当,实时超声造影更加方便灵活,为背驼式肝移植术后静脉并发症的诊断提供了一种可靠的影像学新方法。     

The 5-time point oral glucose tolerance test as a predictor of new-onset diabetes after kidney transplantation

Aims

To evaluate the predictive power of the 5-time point oral glucose tolerance test (OGTT) for new-onset diabetes after kidney transplantation (NODAT).

Methods

We performed a retrospective study of 145 patients without diabetes who received kidney transplantations at our hospital. The 5-time point OGTT was performed before transplantation. The area under a receiver-operating characteristic curve (aROC) was used for evaluating the predictive power of 5-time point OGTT values.

Results

Seventeen patients developed NODAT within 1 year after transplantation. All postload plasma glucose (PPG) levels were higher in patients who developed NODAT than in those who did not; fasting plasma glucose levels were not different. The aROC for the area under the glucose concentration-time curve was significantly greater than that for fasting plasma glucose. Univariate and multivariate analyses showed that each PPG level was an independent risk factor for NODAT. Furthermore, patients with normal glucose tolerance (NGT) or impaired glucose tolerance (IGT) could be stratified with a 1-h plasma glucose (1h-PG) cut-off point of 8.4 mmol/L. The incidences of NODAT were 23.5%, 16.7%, 9.1%, and 0% for patients with IGT + 1h-PG ≥8.4 mmol/L,IGT + 1h-PG <8.4 mmol/L, NGT + 1h-PG ≥ 8.4 mmol/L, and NGT + 1h-PG < 8.4 mmol/L, respectively.

Conclusions

The area under the glucose concentration-time curve and each PPG concentration during the 5-time point OGTT are strong predictors of NODAT. A 1h-PG cut-off point of 8.4 mmol/L plus NGT/IGT can be used to identify patients at intermediate and high risk of developing NODAT.     

Physician attitudes toward the use of fecal microbiota transplantation for the treatment of recurrent Clostridium difficile infection

BACKGROUND:

Fecal microbiota transplantation (FMT) is a safe and effective, yet infrequently used therapy for recurrent Clostridium difficile infection (CDI).

OBJECTIVE:

To characterize barriers to FMT adoption by surveying physicians about their experiences and attitudes toward the use of FMT.

METHODS:

An electronic survey was distributed to physicians to assess their experience with CDI and attitudes toward FMT.

RESULTS:

A total of 139 surveys were sent and 135 were completed, yielding a response rate of 97%. Twenty-five (20%) physicians had treated a patient with FMT, 10 (8%) offered to treat with FMT, nine (7%) referred a patient to receive FMT, and 83 (65%) had neither offered nor referred a patient for FMT. Physicians who had experience with FMT (performed, offered or referred) were more likely to be male, an infectious diseases specialist, >40 years of age, fellowship trained and practicing in an urban setting. The most common reasons for not offering or referring a patient for FMT were: not having ‘the right clinical situation’ (33%); the belief that patients would find it too unappealing (24%); and institutional or logistical barriers (23%). Only 8% of physicians predicted that the majority of patients would opt for FMT if given the option. Physicians predicted that patients would find all aspects of the FMT process more unappealing than they would as providers.

CONCLUSIONS:

Physicians have limited experience with FMT despite having treated patients with multiple recurrent CDIs. There is a clear discordance between physician beliefs about FMT and patient willingness to accept FMT as a treatment for recurrent CDI.     

Platelet‐Rich Plasma–Assisted Guided Bone Regeneration for Ridge Augmentation: A Randomized,Controlled Clinical Trial

Background: Platelet‐rich plasma (PRP) contains a number of biologically active growth factors, and previous studies have reported conflicting ridge augmentation results. The primary aim of this randomized, controlled, masked, clinical trial was to determine if PRP combined with a rapidly resorbing cancellous allograft would enhance the regenerative result compared with an allograft without PRP. Methods: Thirty‐two patients with an edentulous ridge defect were sequentially entered into the study; four were excluded from data analysis. Fourteen patients received a cancellous allograft (CAN group) and the other 14 received a cancellous allograft mixed with PRP (PRP group). All 28 grafted sites were covered with a resorbable polylactide membrane. After elevation of a full‐thickness flap, horizontal ridge dimensions were measured with a digital caliper at the crest and 5 mm apical to the crest. Vertical ridge dimensions were measured from a tooth‐supported stent. All sites were reentered at 4 months, and a trephine core was obtained for histologic analysis before implant placement. Results: The crestal ridge width for the CAN group had a mean gain of 2.0 ± 1.2 mm, whereas the PRP group gained 2.9 ± 1.0, and the difference was statistically significant between groups (P <0.05). The percent vital bone was 36% ± 14% for the CAN group compared with 51% ± 15% for the PRP group and was statistically significant between groups (P <0.05). Loss of augmented ridge width was 34% ± 17% for the CAN group and 28% ± 17% for the PRP group (P >0.05). Conclusion: These clinical and histologic findings suggest that PRP enhanced bone regeneration and resulted in increased horizontal bone gain and percentage vital bone.