The different degrees of lymphocytic infiltration observed in Graves' disease, Hashimoto's disease, and De Quervain thyroiditis suggest that the regulation of adhesion molecules expressed on endothelial cells could be different in these autoimmune disorders of the thyroid. Using immunohistological techniques, we observed that thyroid samples from patients with Graves' disease displayed a characteristic pattern of capillary proliferation, with CD62P, CD62E, and CD31 expression on endothelial cells. This was different from the pattern and size of endothelial cells expressing adhesion molecules in the two other types of thyroiditis where larger vessels and high endothelial venules were stained. Almost no signs of endothelial cell activation could be seen in a comparative series of non-autoimmune disorders.

Introduction: Evidence has accumulated for the role of endothelial damage in systemic sclerosis (SSc) and the anti-endothelial cell antibodies (AECAs) might underlie vascular injury.

Aim: Since endothelial microparticles (EMPs) and circulating endothelial cells (CECs) reflect endothelial damage, we aimed to investigate their possible relationship with AECAs in SSc. We examined whether AECAs could affect endothelial repair based on the number of endothelial progenitor cells (EPCs).

Material and methods: Forty-seven SSc patients were screened. The AECAs were identified in serum by indirect immunofluorescence. EPCs and CECs were isolated from the peripheral blood using anti-CD34-based immunomagnetic separation, whereas EMPs were analyzed in plasma. Flow cytometry was used to quantify EMPs, CECs and EPCs.

Results: AECAs were found in 21 (44.7%) SSc patients and were significantly associated with higher levels of total as well as apoptotic (AnnV+ and CD51+) EMPs, whereas activated (CD62E+/AnnV-) EMPs did not differ between groups. Patients with AECAs had significantly elevated total CECs as well as activated CD105+ CECs. Total endothelial progenitors did not differ between patients with or without AECAs; however AECAs was negatively associated with the population of EPCs that express VEGFR2 or Tie2 receptors.

Conclusions: We found an association between AECAs and the severity of endothelial damage in SSc based on higher levels of total EMPs and CECs. In our study, AECAs were associated with apoptosis of ECs rather than their activation. We also identified a possible role of AECAs in the impairment of vascular repair in SSc as evidenced by significantly fewer angiogenic EPCs.

Keywords: anti-endothelial cell antibodies; endothelial cells; endothelial microparticles; systemic sclerosis.

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. β-1,4-Galactosyltransferase I (β-1,4-GalT-I) is a major galactosyltransferase responsible for selectin-ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of β-1,4-GalT-I was low in normal or complete Freund's adjuvant (CFA) control rats' spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of β-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of β-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of β-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65-25 kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that β-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that β-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin-ligand biosynthesis and interaction with E-selectin.

Intense, large muscle mass exercise increases circulating microvesicles, but our understanding of microvesicle dynamics and mechanisms inducing their release remains limited. However, increased vascular shear stress is generally thought to be involved. Here, we manipulated exercise-independent and exercise-dependent shear stress using systemic heat stress with localized single-leg cooling (low shear) followed by single-leg knee extensor exercise with the cooled or heated leg (Study 1, n = 8) and whole-body passive heat stress followed by cycling (Study 2, n = 8). We quantified femoral artery shear rates (SRs) and arterial and venous platelet microvesicles (PMV-CD41+) and endothelial microvesicles (EMV-CD62E+). In Study 1, mild passive heat stress while one leg remained cooled did not affect [microvesicle] (P ≥ 0.05). Single-leg knee extensor exercise increased active leg SRs by ~12-fold and increased arterial and venous [PMVs] by two- to threefold, even in the nonexercising contralateral leg (P < 0.05). In Study 2, moderate whole-body passive heat stress increased arterial [PMV] compared with baseline (mean±SE, from 19.9 ± 1.5 to 35.5 ± 5.4 PMV.μL-1.103, P < 0.05), and cycling with heat stress increased [PMV] further in the venous circulation (from 27.5 ± 2.2 at baseline to 57.5 ± 7.2 PMV.μL-1.103 during cycling with heat stress, P < 0.05), with a tendency for increased appearance of PMV across exercising limbs. Taken together, these findings demonstrate that whole-body heat stress may increase arterial [PMV], and intense exercise engaging either large or small muscle mass promote PMV formation locally and systemically, with no influence upon [EMV]. Local shear stress, however, does not appear to be the major stimulus modulating PMV formation in healthy humans.

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.

OBJECTIVE

There are few reports on the migration of CLA+ T cells through E-selectin in cutaneous lichen planus, with only one study on oral lichen planus (OLP). This study aimed to analyze CLA expression and assess whether there is a correlation with E-selectin (CD62E) in OLP lesions.

METHODS

Biopsies were performed on 11 patients including two areas: one without clinical and histopathological features of OLP [perilesional group (PLG)] and the other with clinical and histopathological features of OLP [OLP group (OLPG)]. The specimens obtained were divided into two: One was fixed in formalin for routine analysis (H&E), and the other was frozen for CD3, CD4, CD8, CLA, and CD62E immunofluorescence markers.

RESULTS

More CD4+ (median 1409, range 860-2519), CD8+ (median 1568, range 654-3258), and CLA+ T cells (median 958, range 453-2198) and higher CD62E expression (median 37, range 27-85) were identified in OLPG (P = 0.003; P = 0.003; P = 0.004; P = 0.003, respectively) than those in PLG. The median prevalence analysis was also significantly higher for CLA+CD8+ T cells in OLPG (OLPG = 39.4%, range 18.4-64.2; PLG = 29.4%, range 12.1-47.1) (P = 0.026). None of the correlations between CD3+ or CLA+ T cells and CD62E in OLPG and in PLG were significant.

CONCLUSIONS

The significant presence of CLA+ T cells and E-selectin expressions in the OLPG suggests their involvement in the etiopathogenesis of OLP; however, only a weak correlation between CLA+ T cells and E-selectin was observed.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (VCAM-1/CD106), induced phosphorylation of AKT and NF-κB in mouse bone marrow-derived macrophages (BMDM) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidyl inositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both IL-10 and CCL2, but not TNF or IL-6 in response to LPS stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDM to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest E-selectin plays an early role in priming and amplifying innate immune responses.

Keywords: AKT; CCL2; E-selectin; adhesion molecules; mTOR; macrophage activation.

Promising biomarkers which may help predict the risk of developing severe dengue virus infection (DVI) are lacking and will be helpful. Thus the main aim of this study was to analyze the role of cell-derived microparticles (MP) in DVI. Sixty patients with DVI i.e. 18: dengue with warning signs (DWS); 1: DSS and 41: dengue without warning signs (DWOS); along with 15 controls (other febrile illness) were included in the study. The following MPs were assessed: annexinV, platelet (CD41a), red blood cell (RBC) (CD235a) and activated endothelial (CD62e) MPs. Patients with profound thrombocytopenia without bleeding had statistically elevated platelet MP (PMP) levels when compared to patients with profound thrombocytopenia with bleeding (p < .001). RBC MPs were found to be significantly elevated in the 2nd phase in patient with DWS which was seen earliest on day 4 of infection with a cut off of ≥2200 MPs/μl when compared to patients with DWOS (p < .0001). PMPs may prove to be a promising novel biomarker which helps discriminate patients in need of prophylactic platelet transfusion from those who do not. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, MPs seem to be a promising important biomarker in many aspects of DVI.

We previously described a subgroup of immune thrombocytopenic purpura (ITP) patients presenting with recurring transient ischemic attack-like symptoms and progressive cognitive impairment due to small vessel disease (SVD) seen in the brain. They presented minimal bleeding despite thrombocytopenia, and platelet activation was elevated compared to classic ITP. On the hypothesis that the blood-brain barrier (BBB) is compromised in this subgroup, we investigated the effect of plasma from SVD-ITP patients on the transendothelial migration of leukocytes (TEML). Brain microvascular endothelial cells (BMVEC) were grown to confluence on 6.5-microm pore filters and plasma from 10 healthy controls, 20 classic ITP, and 5 SVD-ITP were added and incubated 24 hr. Then 1 x 10(5) monocytes (U937) were added and the number migrated through the EC monolayer after 6 hr was measured by flow cytometry. The effect on TEML of danazol was also assessed. We found that plasma from SVD-ITP but not classic ITP induced 10-fold rise in EC activation marker CD62E and a sevenfold increase in TEML, to 38.5% +/- 12.5% of cells migrated, compared to normal controls (5.6% +/- 1.2%) or classic ITP (6.1% +/- 0.2%), P < 0.001. Preincubation of U937 with endothelial microparticles (EMP) increased TEML by 20.0% +/- 6.4% with SVD-ITP plasma, significantly more than with classic ITP or control plasmas, P = 0.003. Pretreatment of cultures with danazol (100 microg/mL) inhibited TEML by 25% in all wells tested, whether or not EMP were added. In summary, SVD-ITP plasma activates EC and augments TEML, suggesting plasma-mediated BBB dysfunction in this syndrome. Danazol modestly but significantly inhibited TEML.

Kinetic and phenotypic heterogeneity in leukocyte subsets and adhesion-molecule expression is characteristic of many inflammatory conditions. We have studied the effect of various cytokines and inflammatory agonists on the type of leukocyte present and the adhesion molecules expressed in acute lesions (up to 3 days old) in porcine skin by immunohistology. Four major histocompatability complex-homozygous inbred pigs received replicate intradermal injections of IL-1 alpha, TNF-alpha, PMA, or PHA. Injections were timed so that lesions were obtained at 2, 4, 9, and 24 hours. Another two animals were studied at time points up to 72 hours. Leukocyte subsets and endothelial adhesion molecules were visualized on cryosections by use of monoclonal antibodies and alkaline phosphatase immunohistologic techniques. Substantial heterogeneity in leukocyte phenotypes was observed with all agonists, with lymphocyte subsets showing the greatest variation. Thus, CD2+ and gamma delta TcR+ (Null) T lymphocytes were present in all lesions, but to a varying extent such that few T cells were seen after PMA injection, approximately equal proportions of each after TNF-alpha, but substantially more gamma delta TcR+ (Null) lymphocytes were noted after PHA administration. Endothelial adhesion molecules were also variously affected, with E-selectin (CD62E) being transiently up-regulated by IL-1 alpha but CD62E showed early and sustained expression after TNF-alpha and PHA administration. The E-selectin ligand was demonstrated on infiltrating gamma delta TcR+ lymphocytes by use of a recombinant porcine E-selectin. The L-selectin ligand, identified by the mAb MECA-79, was only observed in late acute sites (< 24 hours) of TNF-alpha and PHA. Endothelial expression of class II major histocompatability complex was also variously up-regulated by all agonists. The results underline the heterogeneity of leukocyte phenotypes and endothelial adhesion molecule expression in acute cutaneous lesions dependent upon the nature of the inflammatory agonist and indicate an association between endothelial E-selectin and the presence of a gamma delta TcR+ (Null) T lymphocyte subset.

A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV with scanned areas of 200 x 400 microns. The semi-quantitative data indicated that in LPS-treated groups both adhesion molecules were expressed at a significantly higher level than in all other groups (P < 0.01). In addition, after a 4 h treatment the expression levels of E-selectin in all groups were higher compared to ICAM-1. The experimental data from X-ray microanalysis were compared with data obtained from an enzyme-linked immunosorbent assay (ELISA) and similar values were found for both types of preparation. This microanalytical method is relatively simple and seems to be suitable for immunogold labelling studies on different types of endothelial cells in vitro.

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLe^X), displayed on circulating cells. Though CAR T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLe^X and do not bind E-selectin. However, enforced sLe^X display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom-installation of sLe^X programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.

Cocoa flavanols (CFs) can improve flow-mediated dilation (FMD), blood pressure, and vascular stiffness in healthy subjects. Endothelial microparticles (EMPs) are markers of endothelial functional integrity, reflecting activation and injury. In plasma samples, we investigated whether age-dependent changes in circulating EMPs exist and whether CFs decrease EMPs in healthy humans. The concentrations of CD31⁺/41^-, CD144⁺, and CD62e⁺ EMPs (flow cytometry) were increased in healthy elderly (n = 19) compared to young (n = 20) non-smokers. EMPs correlated with age, systolic blood pressure, and pulse wave velocity. CD31⁺/41^- and CD62e⁺ EMPs inversely correlated with FMD. Following 2 weeks twice-daily CF consumption (450 mg), CD31⁺/41^- and CD144⁺ EMPs decreased in both young and elderly subjects compared to the CF-free control. The EMP decrease inversely correlated with FMD improvements. Cardiovascular aging is associated with increased EMPs that can be modulated by dietary flavanols along with improvements in vascular function. This indicates that flavanol consumption can improve endothelial functional integrity in healthy humans.

OBJECTIVE

Systemic inflammation, measured by C-reactive protein (CRP), is an important risk factor for cardiovascular disease (CVD) and mortality. We investigated whether aerobic exercise training (AEXT) affects African Americans with high inflammation (HI) the same way it does African Americans with low inflammation (LI) in terms of CVD risk factors.

METHODS

23 African Americans with CRP levels <3 mg/L (LI) and 14 African Americans with CRP ≥3 mg/L (HI) underwent six months of AEXT. Participants were sedentary, non-diabetic, non-smoking, with clinical blood pressure <160/100 mm Hg, were non-hyperlipidemic, had no signs of cardiovascular, renal, or pulmonary disease, and were not on medication. Measures included CD62E+ endothelial microparticles (EMPs), a measure of early stage endothelial dysfunction, as well as lipid and glucose profile, aerobic fitness, body composition, and blood pressure.

RESULTS

The LI group improved aerobic fitness by 10%, body mass index by 3%, and plasma triglycerides by 20%, with no change being observed in HI group for these variables. The HI group improved fasting plasma glucose levels by 10%, with no change occurring in the LI group. Both groups improved CD62E+ EMPs by 38% and 59% for the LI and HI group, respectively.

CONCLUSIONS

A standard AEXT intervention differentially affected CVD risk factors among African Americans with high and low inflammation. This may indicate that, in African Americans with high inflammation, AEXT alone may not be enough to reap the same benefits as their low-inflammation peers in terms of CVD risk modification.

Objective: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients.

Methods: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF⁺MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations.

Results: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF⁺MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF⁺MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF⁺MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF⁺MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05).

Conclusion: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF⁺MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

题目: ET患者血浆MP水平测定及其与JAK2V617F突变关系的研究.

目的: 检测原发性血小板增多症（ET）患者血浆中的微颗粒（MP）水平，并分析其与JAK2V617F突变的关系.

方法: 抽取56例ET患者及28例正常人空腹静脉血，枸橼酸钠抗凝(1∶9)，以藻红蛋白标记的特异性荧光抗体CD235a-PE、CD61-PE、CD142-PE和CD62E-PE 分别标记红细胞微颗粒（RMP）、血小板微颗粒（PMP）、组织因子微颗粒（TF⁺MP）及内皮细胞微颗粒（EMP），FCM检测4种MP绝对数。试剂盒抽提基因组DNA，PCR扩增目标DNA，琼脂糖凝胶电泳筛选26例JAK2V617F突变标本.

结果: ET组的RMP（157.2±304.9/μl vs 21.3±18.4/μl）、PMP（1378.9±2454/μl vs 113.8±97.1/μl）、TF⁺MP（123±354.6/μl vs 21±15.7/μl）、EMP（1434.7±2601.9/μl vs 291.7±297.7/μl）高于正常对照组（P<0.05）；8例血栓组的RMP（258.2±327.7/μl vs 55.6±63.8/μl）、PMP（1853±1874.7/μl vs 444.7±712/μl）、TF⁺MP（120.4±112.5/μl vs 60.3±128.3/μl）、EMP（1637.1±1563.5/μl vs 629.4±1000.2/μl）高于25例非血栓组（P<0.05）；17例脾肿大组的RMP（306.8±491.1/μl vs 59.3±51/μl）、PMP（2944.8±3767.6/μl vs 334.8±420.2/μl）、TF⁺MP（162.9±166.8/μl vs 31.0±28.7/μl）、EMP（3031.8±4048.8/μl vs 701.5±729.2/μl）高于11例非脾肿大组（P<0.05）；26例JAK2V617F突变组的TF⁺MP高于未突变组（154.7±516.3/μl vs 100.5±126.6/μl）（P<0.05），另外3种MP在这2组间无明显区别（P>0.05）.

结论: ET组、血栓组、脾肿大组4种MP水平均高于对照组；JAK2V617F突变组患者TF⁺MP数高于未突变组，另3种MP水平在突变与未突变组之间无明显区别.

BACKGROUND

Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker.

OBJECTIVE

This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes.

METHODS

Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion.

RESULTS

Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood.

CONCLUSIONS

Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis.

BACKGROUND

Rolling of leukocytes at the surface of the vascular endothelium is a prerequisite for a subsequent firm adhesion, particularly the slow rolling appearing on ELAM CD62E. Therefore, it may be considered that increasing the rolling velocities should be a precise therapeutic target in clinical situations where leukocytes accumulate, mainly venous and arterial ischaemia.

METHODS

Human neutrophils were allowed to flow on endothelial HUVECs, with and without 4 hours interleukin-1alpha activation, the cells having or not been incubated with INO5042 anti-inflammatory drug. Under a mean shear-stress of 2 dyn/cm(2), rollers and stickers were identified and quantified, using a video-camera and picture analysing software.

RESULTS

When the drug had been added to endothelial cells a shift of velocities was observed towards fast speeds (from 3-5 to 7-11 microm/sec). The same results was significantly found when neutrophils, alone or along with endothelium, had been submitted to the drug, the number of stickers and rollers beeing reduced as well. Finally, such a precise pharmacological method proved efficient to detect the exact mechanism of INO5042 on white cell adhesion.

OBJECTIVE

To characterize the immunohistopathological features of oral chronic graft-versus-host disease (cGVHD), and the impact of topical immunomodulatory therapy on the infiltrating cells.

METHODS

Paired oral cGVHD biopsies obtained before (n = 12) and 1 month after treatment (n = 12) with topical dexamethasone (n = 8) or tacrolimus (n = 4) were characterized by immunohistochemistry using a panel of CD1a, CD3, CD4, CD8, CD20, CD31, CD62E, CD103, CD163, c-kit, and FoxP3. Controls included acute GVHD (aGVHD; n = 3), oral lichen planus (OLP; n = 5), and normal tissues (n = 5).

RESULTS

Oral cGVHD specimens prior to treatment were mainly characterized by basal cell squamatization, lichenoid inflammation, sclerosis, apoptosis, and lymphocytic exocytosis. The infiltrating cells in oral cGVHD primarily consisted of CD3+ , CD4+ , CD8+ , CD103+ , CD163+ , and FoxP3+ cells, which were higher than in normal tissues. Topical dexamethasone or tacrolimus reduced neutrophilic exocytosis, basal cell squamatization, and lichenoid inflammation in oral cGVHD, and dexamethasone reduced the number of CD4+ and CD103+ cells.

CONCLUSIONS

The high expression of CD3, CD4, CD8, CD103, CD163, and FoxP3 confirms that oral cGVHD is largely T-cell-driven with macrophage participation. The impact of topical immunomodulatory therapy was variable, reducing histological inflammatory features, but with a weak clinicopathological correlation. Topical dexamethasone reduced the expression of CD4 and CD103, which may offer novel therapeutic targets.

BACKGROUND

Regarding recent findings on the proven influence of laser light on healing and endothelial regeneration processes, this study was conducted in order to examine the influence of low-power laser illumination on the endothelial inflammatory response.

METHODS

Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cord, cultured in standard conditions, were harvested and passaged in 24-well plates. Laser influence on HUVEC inflammatory response was measured by stimulating them with Interleukin-1β (IL-1β) followed by laser light illumination. A 808-nm wave length laser diode and light energy doses of 1.5 and 4.5 J/cm(2) were used (50 mW for 90 and 270 s respectively). The response was measured by assessing Cluster of differentiation 54 (CD54), Cluster of differentiation 62E (CD62E), Monocyte chemotactic protein-1 (MCP-1) expression and von Willebrand factor (vWF) release, at 6 and 24 h after stimulation. The MCP-1 and vWF activity in cell supernatants was measured with an enzyme-linked immunosorbent assay (ELISA) kit. Cytofluorometry was used to assess CD54 and CD62E expression.

RESULTS

MCP-1 concentration in supernatants from HUVECs was significantly lower 6 h after 4.5 J/cm(2) stimulation compared to IL-1β-stimulated cells. No changes in MCP-1 levels after IL-1β stimulation plus 1.5 J/cm(2) illumination, compared to IL-1β stimulated HUVECs were noted. IL-1β stimulation significantly enhanced the concentration of vWF and the expression of CD54 and CD62E. Both energies of laser light illumination inhibited the IL-1β-induced increase of CD54 and CD62E concentration. vWF activity after illumination was comparable to that of unstimulated cells. There were no significant differences in the viable cell count between the groups tested.

CONCLUSIONS

Low-power laser illumination diminishes the pro-inflammatory and pro-coagulant activity of IL-1β-stimulated HUVECs.