OBJECTIVE

Growing evidence suggests that endothelial injury is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). Circulating endothelial microparticles (EMPs) increase in patients with COPD because of the presence of endothelial injury. We examined the relationship between EMP number and changes in forced expiratory volume in 1 s (FEV1) in patients with COPD.

METHODS

Prospective study.

METHODS

One hospital in Japan.

METHODS

A total 48 outpatients with stable COPD coming to the hospital from September 2010 to September 2011.

UNASSIGNED

Blood samples were collected and vascular endothelial (VE)-cadherin EMPs (CD144+ EMPs), E-selectin EMPs (CD62E+ EMPs) and platelet endothelial cell adhesion molecule EMPs (CD31+/CD41- EMPs) were measured using fluorescence-activated cell sorting. Annual FEV1 changes were evaluated using FEV1 data acquired a year before and a year after sample collection.

RESULTS

The number of E-selectin and VE-cadherin EMPs showed significant negative correlations with annual FEV1 changes (rs=-0.65, p<0.001, rs=-0.43, p=0.003, respectively). Leucocyte counts tended to be correlated with annual FEV1 changes, but this correlation was not significant (rs=-0.28, p=0.057). There were significant differences in annual FEV1 changes between with and without history of frequent exacerbation (p=0.006), and among Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages (p=0.009). Multiple linear regression analysis revealed E-selectin EMP to be the only significant parameter associated with annual FEV1 changes, independent of VE-cadherin EMP, GOLD stages, leucocyte counts, and history of frequent exacerbation. Receiver operating characteristic curves showed the optimum E-selectin EMP cut-off level for prediction of rapid FEV1 decline (>66 mL/year) to be 153.0/µL (areas under curve 0.78 (95% CI 0.60 to 0.89); sensitivity, 67%; specificity, 81%).

CONCLUSIONS

The high E-selectin EMP levels in stable patients with COPD are predictive of rapid FEV1 decline.

BACKGROUND

UMIN000005168.

Many preclinical studies in investigative dermatology are performed preferably in pigs because pig skin is more similar to human skin than is rodent skin. A frequently used model is allergic contact dermatitis (ACD); however, this T-cell-mediated skin condition so far is not well characterized in pigs. The present study is aimed at the evaluation of morphologic and immunohistochemical features of experimentally induced acute ACD in Göttingen minipigs using 2,4-dinitrofluorobenzene (DNFB) as a hapten. Eight minipigs were sensitized with 10% DNFB and challenged 2 weeks later at different sites with 1% DNFB. In addition to clinical examinations, cutaneous blood flow was quantified by laser Doppler velocimetry (Periflux PF3). These examinations were performed before challenge and 8, 24, 48, and 72 hours after challenge. Skin biopsies were taken at the same time points, fixed, sectioned, and stained with Giemsa for histologic evaluation, or with mouse anti-swine monoclonal antibodies (CD1, CD2, CD4, CD5, CD8, CD25, CD45, MHCII) and with one mouse anti-human monoclonal antibody (CD62E) cross-reacting with swine for immunohistochemical evaluation. Positively stained cells were counted per square millimeter of epidermis and dermis by using a video image analyzing system (Videoplan Kontron). Erythema and cutaneous blood flow peaked at 24 hours. The major epidermal changes most pronounced at 48 hours were acanthosis, spongiosis, intracellular edema, exocytosis, and abscesses mainly containing neutrophils and mononuclear cells (MNC). Perivascular infiltrates of MNC as well as neutrophils and eosinophils were the most significant dermal changes, with peak levels at 24-48 hours. In biopsies taken before challenge, CD1+ dendritic cells were found in similar numbers and locations as MHCII+ cells in the epidermis. In the epidermis the maximum CD1+ cell decrease occurred at 24 hours whereas in the dermis the maximum increase in CD1+ stained cells was seen at 72 hours. The dermal infiltrate (CD2+, CD5+, CD25+, and CD45+) was most dense at 48 hours. Between 8 and 48 hours more CD4+ were present than CD8+, cells, whereas at 72 hours CD4+ and CD8+ cells were similar in numbers. These findings closely resemble changes in human ACD. Therefore, DNFB-induced ACD in Göttingen minipigs is considered to be an appropriate animal model to study immunopathologic mechanisms and pharmacologic intervention.

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real-time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM-1 (CD54) and E-selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3⁺ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF-α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8⁺ cells were detected in TNF-α-treated melanoma metastases compared with PBS-treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI-67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.

Mucosal Leishmaniasis (ML) may occur in both nasal and oral mucosa. However, despite the impressive tissue destruction, little is known about the oral involvement. To compare some changes underlying inflammation in oral and nasal ML, we performed immunohistochemistry on mucosal tissue of 20 patients with ML (nasal [n = 12]; oral [n = 8] lesions) and 20 healthy donors using antibodies that recognize inflammatory markers (CD3, CD4, CD8, CD22, CD68, neutrophil elastase, CD1a, CLA, Ki67, Bcl-2, NOS2, CD62E, Fas and FasL). A significantly larger number of cells, mainly T cells and macrophages, were observed in lesions than in healthy tissue. In addition, high nitric oxide synthase 2 (NOS2) expression was associated with a reduced detection of parasites, highlighting the importance of NOS2 for parasite elimination. Oral lesions had higher numbers of neutrophils, parasites, proliferating cells and NOS2 than nasal lesions. These findings, together with the shorter duration of oral lesions and more intense symptoms, suggest a more recent inflammatory process. It could be explained by lesion-induced oral cavity changes that lead to eating difficulties and social stigma. In addition, the frequent poor tooth conservation and gingival inflammation tend to amplify tissue destruction and symptoms and may impair and confuse the correct diagnosis, thus delaying the onset of specific treatment.

Extracellular vesicles (EVs) are a heterogeneous group of actively released vesicles originating from a wide range of cell types. Characterization of these EVs and their proteomes in the human plasma provides a novel approach in clinical diagnostics, as they reflect physiological and pathological states. However, EV isolation is technically challenging with the current methods having several disadvantages, requiring large sample volumes, and resulting in loss of sample and EV integrity. Here, we use an alternative, non-contact method based on a microscale acoustic standing wave technology. Improved coupling of the acoustic resonator increased the EV recovery from 30% in earlier reports to 80%, also displaying long term stability between experiment days. We report a pilot study, with 20 subjects who underwent physical exercise. Plasma samples were obtained before and 1 h after the workout. Acoustic trapping was compared to a standard high-speed centrifugation protocol, and the method was validated by flow cytometry (FCM). To monitor the device stability, the pooled frozen plasma from volunteers was used as an internal control. A key finding from the FCM analysis was a decrease in CD62E+ (E-selectin) EVs 1 h after exercise that was consistent for both methods. Furthermore, we report the first data that analyse differential EV protein expression before and after physical exercise. Olink-based proteomic analysis showed 54 significantly changed proteins in the EV fraction in response to physical exercise, whereas the EV-free plasma proteome only displayed four differentially regulated proteins, thus underlining an important role of these vesicles in cellular communication, and their potential as plasma derived biomarkers. We conclude that acoustic trapping offers a fast and efficient method comparable with high-speed centrifugation protocols. Further, it has the advantage of using smaller sample volumes (12.5 μL) and rapid contact-free separation with higher yield, and can thus pave the way for future clinical EV-based diagnostics.

BACKGROUND

Neutrophils participate in acute vascular rejection (AVR) of organ xenografts. Induced antibodies (Abs), including anti-Galalpha1,3Gal (alpha-Gal) Abs, have been suggested to cause AVR. We investigated the adhesion of naive human neutrophils to porcine aortic endothelial cells (PAECs) stimulated with anti-alpha-Gal Abs under conditions of flow. In addition, the ability of human neutrophils to adhere to human and porcine endothelium under static and flow conditions was evaluated.

RESULTS

In a flow-adhesion assay, a significant increase in adhesion of human neutrophils to PAECs, but not to human aortic endothelial cells (HAECs), was detected 6 hours after anti-alpha-Gal Ab-binding. After Ab stimulation, PAECs expressed CD62E and increased levels of CD106, indicating an activated endothelial cell (EC) phenotype. In a migration assay, supernatants from Ab-stimulated PAECs induced migration of human neutrophils, which was partially blocked by anti-porcine (p) interleukin (IL)-8 Abs and an antagonist to platelet-activating factor (PAF). In static and flow-adhesion assays, no difference in adhesion of human neutrophils to unstimulated or tumor necrosis factor (TNF)-alpha-stimulated HAECs and PAECs could be detected.

CONCLUSIONS

Our data suggest that anti-alpha-Gal Abs play an important role in the initiation of AVR by mediating adhesion and recruitment of neutrophils within an organ xenograft. In contrast with previous investigations, our data argues against a differential recognition of PAECs and HAECs by human neutrophils. Thus, to prevent AVR and accomplish long-term xenograft survival, it will be important to remove anti-alpha-Gal Abs before and after pig-to-human transplantation.

Cytokine mediators and leukocyte-endothelial cell adhesion molecules are critical and interdependent components of the acute inflammatory response in sepsis. We hypothesized that the administration of monoclonal antibodies to intercellular adhesion molecule-1 (CD54) or E- and L-selectin (CD62E/L) would decrease serum levels of the proinflammatory cytokines interleukin-1beta (IL-1), IL-6, and IL-8 and tumor necrosis factor receptor (TNFR-1) in baboons during sepsis. Adult male baboons received infusions of 1 x 10(9) colony forming units (CFU)/kg heat-killed Escherichia coli (E. coli) followed 12 h later by live E. coli (1 x 10(10) CFU/kg). At the time of live bacterial infusion, six septic animals were treated with a monoclonal antibody to CD54 and six with an antibody to CD62E and L (1 mg/kg). Eight untreated septic animals served as controls. Sequentially drawn serum samples were assayed for IL-1, IL-6, IL-8, and TNFR-1 using enzyme-linked immunoassay (ELISA). Data were compared using Mann-Whitney U tests and Chi-square analyses. Median survival was decreased in both treatment groups compared to controls (P < 0.05). Peak IL-1 level was higher than controls in septic animals treated with anti-CD54 but not anti-CD62E/L (P < 0.05, P = NS, respectively). Elevations in IL-6, IL-8, and TNFR-1 were increased and prolonged in both antibody treated groups compared to controls (P < 0.05). These results provide the first in vivo evidence that leukocyte-endothelial adhesion molecules CD54 and CD62E/L regulate cytokine production in sepsis.

The effect of endurance exercise on circulating microvesicle dynamics and their impact on surrounding endothelial cells is unclear. Here we tested the hypothesis that exercise intensity modulates the time course of platelet (PMV) and endothelial-derived (EMV) microvesicle appearance in the circulation through hemodynamic and biochemical-related mechanisms, and that microvesicles formed during exercise would stimulate endothelial angiogenesis in vitro. Nine healthy young men had venous blood samples taken before, during, and throughout the recovery period after 1 h of moderate [46 ± 2% maximal oxygen uptake (V̇o2max)] or heavy (67 ± 2% V̇o2max) intensity semirecumbent cycling and a time-matched resting control trial. In vitro experiments were performed by incubating endothelial cells with rest and exercise-derived microvesicles to examine their effects on cell angiogenic capacities. PMVs (CD41+) increased from baseline only during heavy exercise (from 21 ± 1 × 103 to 55 ± 8 × 103 and 48 ± 6 × 103 PMV/μl at 30 and 60 min, respectively; P < 0.05), returning to baseline early in postexercise recovery (P>> 0.05), whereas EMVs (CD62E+) were unchanged (P>> 0.05). PMVs were related to brachial artery shear rate (r2 = 0.43) and plasma norepinephrine concentrations (r2 = 0.21) during exercise (P < 0.05). Exercise-derived microvesicles enhanced endothelial proliferation, migration, and tubule formation compared with rest microvesicles (P < 0.05). These results demonstrate substantial increases in circulating PMVs during heavy exercise and that exercise-derived microvesicles stimulate human endothelial cells by enhancing angiogenesis and proliferation. This involvement of microvesicles may be considered a novel mechanism through which exercise mediates vascular healing and adaptation.

OBJECTIVE

To appreciate the evolution of serum angiogenic and/or adhesion molecules levels during a long term follow-up of rheumatoid arthritis (RA) patients.

METHODS

Serum levels of 5 soluble adhesion/angiogenesis glycoproteins (VEGF, CD31, CD54, CD62E, CD106) were measured in Elisa in samples collected over 6 years in a cohort of 43 RA patients with monitored clinical parameters of disease activity and severity.

RESULTS

RA patients had significantly higher levels (p < 0.0001) of sCD106 (VCAM-1) than control subjects. Conversely, the levels of soluble VEGF, CD31, CD54 and CD62E were normal or lower than normal. No statistically significant time effect was noted. No effect either was noted as related to the therapeutic agents taken by the patients.

CONCLUSIONS

The sustained elevated serum levels of sCD106 observed here imply that this molecule might be related to the chronicity and progression of RA.

OBJECTIVE

Rheumatoid arthritis is a chronic inflammatory autoimmune disease with proinflammatory cytokines involved in its pathogenesis. Recently in vitro as well as in vivo studies have shown that iloprost, a stable prostacyclin analogue, can reduce the release of these cytokines. This study was performed to further investigate the anti-inflammatory effects of iloprost by determining plasma adhesion molecules as markers of endothelial cell activation, and plasma thrombomodulin as a parameter of endothelial cell injury in patients with rheumatoid arthritis receiving oral iloprost therapy.

METHODS

Plasma thrombomodulin levels and the values of the plasma adhesion molecules VCAM-1 (vascular cell adhesion molecule 1), E-selectin (CD62E), and ICAM-1 (intercellular adhesion molecule 1, CD 54) were measured by ELISA during a 7-day period of treatment with orally-administered iloprost in 14 patients with active rheumatoid arthritis. Finally, the same parameters were determined at the end of the observation period (1 week after the end of therapy). In addition, the disease activity was measured using the DAS (disease activity score) as well as the patients' self-assessed pain severity, and correlated with the changes of plasma adhesion molecule and thrombomodulin levels.

RESULTS

The plasma levels of all three adhesion molecules as well as of thrombomodulin significantly decreased under therapy with oral iloprost. After 1 week (day 7 of therapy), the mean percent changes from day 0 were -20.1% for VCAM-1 (p = 0.008), -21.2 for ICAM-1 (p = 0.003), -24.6% for E-selectin (p = 0.001), and -21.7% for thrombomodulin (p = 0.003). This decrease lasted up to 1 week after the end of therapy in the case of VCAM-1 (p = 0.023) and ICAM-1 (p = 0.001). Further analysis of the results revealed additional significant correlations between different parameters of clinical disease activity, thrombomodulin and adhesion molecules.

CONCLUSIONS

This study showed hints towards clinical effects in patients with rheumatoid arthritis receiving oral iloprost therapy. Pathophysiologically, the decrease of adhesion molecules points at an immunomodulating effect of iloprost. The observed thrombomodulin-lowering effect of iloprost may indicate stabilisation of endothelial cell function by diminishing endothelial cell injury.

BACKGROUND

Microparticles (MP) are small membrane bound cellular particles that play an important role in thrombosis. This study was carried out to evaluate if increased numbers or procoagulant potential of circulating MP contribute to the heterogeneity in occurrence of thrombosis in heterozygotes carrying Factor V Leiden (FVL) mutation.

METHODS

Levels of circulating platelet (CD41a), endothelial (CD62e) as well as leukocyte (CD45) derived MP from 45 FVL heterozygous individuals were enumerated by flow cytometry and compared with normal controls. Functional studies included enzyme linked immunoassay based prothrombinase activity (ELISA) and modified dilute Russell Viper venom test (DRVVT).

RESULTS

Circulating MP were significantly higher in the FVL cohort compared to the controls (median=2100 vs. 1508 MP/microl, respectively p=0.0021).All subsets of MP (platelet, endothelial and leukocyte) were significantly elevated in the FVL group, the most striking disparity seen in the number of CD45 positive leukocyte MP. Despite the differences in the number of MP between the controls and FVL cohorts, there was no significant difference in the prothrombinase activity recorded by the ELISA (2.0 vs 2.4 PS equivalents; p=0.7374) or clotting time assessed by the DRVVT (47 vs 46 sec, p=0.8118). When the FVL cohort was considered alone there was no significant difference in MP parameters between FVL subjects with or without a history of thrombosis.

CONCLUSIONS

This is the first study on circulating MP levels in subjects who are heterozygote for factor V Leiden. We report that circulating platelet and leukocyte MP are elevated in carriers of this mutation and may be important contributors to risk of thrombosis.

Degenerative aortic stenosis (AS) is the most frequent form of acquired valvular heart disease. AS is known to entail endothelial dysfunction caused by increased mechanical shear stress leading to elevated circulatory levels of microparticles. Endothelial and platelet microparticles (EMP and PMP) are small vesicles that originate from activated cells and thrombocytes. We sought to evaluate whether transcatheter aortic valve implantation (TAVI) procedure would elicit effects on circulating EMP and PMP. 92 patients undergoing TAVI procedure for severe AS were included in this study. Samples were obtained at each visit before TAVI, 1 week post-procedure and at 1, 3 and after 6 months after TAVI and were evaluated using flow cytometry. A 12 month clinical follow-up was also performed. CD62E+ EMP concentration before TAVI was 21.11 % (±6.6 % SD) and declined to 20.99 % (±6.8 % SD) after 1 week, to 16.63 % (±5.4 % SD, p < 0.0001) after 1 month, to 17.08 % (±4.6 % SD, p < 0.0001) after 3 months and to 15.94 % (±5.4 % SD, p < 0.0001) after 6 months. CD31+/CD42b-, CD31+/Annexin+/- EMP remained unchanged. CD31+/CD41b+ PMP evidenced a slight, but statistically significant increase after TAVI and remained elevated during the entire follow-up. Apart from a procedure-related improvement in echocardiographic parameters, TAVI procedure led also to a decline in CD62E+ EMP. The reduction in pressure gradients with less hemodynamic shear stress seems also to have beneficially affected endothelial homeostasis.

Timely diagnosis of bacterial infective endocarditis (IE) is crucial, as mortality remains high in this severe bacterial infection, currently without any distinct biological markers. Our goal was to evaluate potential diagnostic biomarkers by reviewing current literature. The MEDLINE, Embase and Scopus databases were searched for articles published from 1980 through June 2015 restricted to English, Norwegian, Danish and Swedish. Eighteen studies qualified, providing a review of the most promising candidates for future studies. Several studies are inconclusive, since they are characterized by using improper control groups. Patients with IE have bacteremia, and control groups should therefore be patients with bacteremia without IE. Based on current research, N-terminal-pro-B-type natriuretic peptide (NT-proBNP) alone or in combination with Cystatin C (Cys C), lipopolysaccharide-binding protein (LBP), troponins, aquaporin-9 (AQP9), S100 calcium binding protein A11 (S100A11), E-selectin (CD62E) and VCAM-1 (CD54) and interleukin-6 (IL-6) are potential biomarkers for future studies.

BACKGROUND

Circulating endothelial cells (CEC) may be a biomarker of vascular injury and pro-thrombotic tendency, while circulating endothelial progenitor cells (CEP) may be an indicator for angiogenesis and vascular remodelling. However, there is not a universally accepted standardized protocol to identify and quantify these cells and its clinical relevancy remains to be established.

OBJECTIVE

To quantify CEC and CEP in patients with venous thromboembolism (VTE) and with myeloproliferative neoplasms (MPN), to characterize the CEC for the expression of activation (CD54, CD62E) and procoagulant (CD142) markers and to investigate whether they correlate with other clinical and laboratory data.

METHODS

Sixteen patients with VTE, 17 patients with MPN and 20 healthy individuals were studied. The CEC and CEP were quantified and characterized in the blood using flow cytometry, and the demographic, clinical and laboratory data were obtained from hospital records.

RESULTS

We found the CEC counts were higher in both patient groups as compared to controls, whereas increased numbers of CEP were found only in patients with MPN. In addition, all disease groups had higher numbers of CD62E+ CEC as compared to controls, whereas only patients with VTE had increased numbers of CD142+ and CD54+ CEC. Moreover, the numbers of total and CD62+ CEC correlated positively with the white blood cells (WBC) counts in both groups of patients, while the numbers of CEP correlated positively with the WBC counts only in patients with MPN. In addition, in patients with VTE a positive correlation was found between the numbers of CD54+ CEC and the antithrombin levels, as well as between the CD142+ CEC counts and the number of thrombotic events.

CONCLUSIONS

Our study suggests that CEC counts may reveal endothelial injury in patients with VTE and MPN and that CEC may express different activation-related phenotypes depending on the disease status.

Endothelial microparticles (EMPs) are membrane-coated vesicles shed from endothelial cells and are considered markers of the endothelial state. It has been shown that total numbers of circulating EMPs are increased in patients with systemic sclerosis (SSc), but their clinical correlations have not yet been investigated in detail. We aimed to assess possible relationships between circulating EMPs and clinical as well as laboratory features among SSc patients with special attention to possible association with alteration in microvascular morphology objectified on nailfold videocapillaroscopy and clinical signs of microvascular complications.

The study included 47 SSc patients and 27 age- and sex-matched healthy controls. EMPs were identified with flow cytometry after staining platelet-poor plasma with combinations of fluorescent cell-specific monoclonal antibodies (anti-CD31, -51, -42b, -62E and Annexin V). The following types of EMPs were evaluated: total EMPs (CD31+/CD42b-), activated EMPs (CD62E+/AnnV-,) and apoptotic EMPs (CD62E+/AnnV+ or CD51+). Clinical evaluation of patients was obtained, including nailfold videocapillaroscopy.

All types of EMPs were significantly elevated in SSc patients as compared with healthy controls. We found significant inverse correlation between severity of skin involvement and values of total EMPs (r=-0.32; p=0.02) and their levels tended to be lower in SSc patients with digital ulcers when compared to those without ischaemic skin lesions (p=0.09). Total EMPs and activated EMPs showed correlations with the number of ramified capillaries (r=-0.40 and r=0.37, respectively, p<0.05 for both). Moreover, total EMPs inversely correlated with the severity of capillary loss (r=-0.35, p<0.05) and their levels were significantly lower in patients with late NVC pattern with respect to those with early microangiopathy (p<0.05). On the other hand, active NVC pattern was characterized by strongly elevated levels of activated EMPs when compared to an early vascular alteration (p<0.05).

Our results suggest that quantity and phenotype of circulating EMPs might indicate on molecular vascular damage with endothelial dysfunction and to reflect progressive loss of capillaries consequencing in microvascular insufficiency in SSc patients.

Increased serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1, CD54) and of soluble E- (CD62E), but not soluble P- (CD62P) and L- (CD62 L) selectins, were detected in Malagasy patients living in an hyperendemic focus of Schistosoma mansoni. Levels of sICAM-1 remained elevated for several months after treatment with praziquantel. Serum levels of ICAM-1, but not of other markers, were significantly correlated with the disease severity, as indicated by ultrasonographical data, and with some circulating fibrosis markers (at least hyaluronic acid). sICAM-1 level may reflect endothelial inflammatory reactions, probably harmful, in the liver and may be useful for monitoring morbidity evolution in schistosomiasis mansoni.

BACKGROUND

In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro.

METHODS

A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNFα. Adherent T cells were determined by flow cytometry.

RESULTS

Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNFα, IFNγ). TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNFα.

CONCLUSIONS

Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes.

BACKGROUND

Migraine has been considered a vascular risk factor especially in young women. Factors predisposing to endothelial damage in migraine are still being debated. The insufficiency of circulating endothelial precursor circulating cells (EPCs) suggested a link between migraine and cardiovascular risk. This research aimed to study a subtype of EPCs, those expressing e-selectin, to assess endothelial activation and, therefore, endothelial dysfunction in migraine.

METHODS

Consecutive headache patients (n = 99) and 35 adjusted controls were recruited. Total EPCs, defined as CD34+/KDR+ cells, and EPC colony-forming units (CFUs) were assayed. We identified as "early" EPCs those CD62E- EPCs, and "late" EPCs, CD62E+, a surrogate marker for endothelial damage. Plasmatic calcitonin-gene related protein (CGRP) and vascular-endothelial growth factor (VEGF) were analyzed.

RESULTS

We did not find differences in the total number of CFUs among clinical groups. Means of total CD34+/KDR+ and "early" EPCs were not significant among clinical groups. Nevertheless, the mean of "late" EPCs was lower (log(10)-transformed mean = 1.715; SD = 0.393) in the control group than in the migraine patients (log(10)-transformed mean = 2.167; SD = 0.685), even after adjustment by VEGF plasma level and other confounding factors. Linear regression analyses disclosed significant predictors for "late" EPCs for controls vs migraine (β = 0.452 SE ± 0.13; p = 0.001). We did not observe differences between migraine with or without aura.

CONCLUSIONS

We observed higher number of activated EPCs in migraine patients than in controls. CD62E+ EPCs might be considered a marker for vascular damage in migraine patients.

Endothelial progenitor cells were isolated from peripheral blood obtained from 32 healthy volunteers without cardiovascular risk factors who ranged in age from 20 to 61 years (mean [+/- SD] age, 34.1 +/- 9.6 years). The fractions of CD34(+) endothelial progenitor cells expressing kinase insert domain receptor-1, CD62E, or CD31 were analyzed with flow cytometry. Correlation analysis demonstrated that there was no significant correlation between subject age and the fraction of circulating CD34(+) mononuclear cells expressing kinase insert domain receptor-1 (P = 0.324; r = -0.180). Similarly, there was no significant correlation between subject age and the fraction of circulating CD34(+) mononuclear cells expressing CD62E (P = 0.496; r = -0.125) or the fraction of circulating CD34(+) mononuclear cells expressing CD31 (P = 0.245; r = -0.212). In conclusion, the experimental results showed that there was no age-related change in the basal level of circulating endothelial progenitor cells in healthy subjects without cardiovascular risk factors.

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.

E-selectin (CD62E), a cell adhesion molecule for most leukocytes, is known to be expressed exclusively on the cytokine-stimulated endothelial cells mainly by inductive activation of NF-kappaB. Using immunohistochemistry and in situ hybridization, we showed that B lymphocytes and plasma cells in the spleens and lymph nodes from nude mice (T-lymphocyte-deficient), but not from SCID mice (T- and B-lymphocyte-deficient), expressed E-selectin prior to cytokine stimulation. The expression of E-selectin was also confirmed on human B lymphocytes isolated from peripheral bloods. The mouse J774A.1 monocytes could adhere to the marginal zones of mouse spleens in an E-selectin Ab inhibitable manner, suggesting the functional activity of the expressed E-selectin. In addition, ARH-77 cells, a cell line derived from human plasma cells, were found to express E-selectin mRNA and protein and to have a NF-kappaB activity for an E-selectin promoter. NF-kappaB antagonists, such as TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone), dexamethasone and a IkappaBalpha mutant plasmid could inhibit both the NF-kappaB activity and the expression of E-selectin. Transfection with an E-selectin promoter-driven reporter gene construct further verified the E-selectin promoter activity in ARH-77 cells. Again, TPCK, dexamethasone, and the IkappaBalpha mutant plasmid could neutralize this activity. These findings suggest that B lymphocytes and plasma cells can express E-selectin, which is functional for monocytic leukocytes, by a mechanism of constitutive activation of NF-kappaB.

BACKGROUND

Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model.

METHODS

Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR.

RESULTS

After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%+/-9.6% [MCMV] vs. 43.9%+/-5.1% [MCMV]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%+/-7.3% [MCMV] vs. 20.2%+/-1.7% [MCMV]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4, CD8, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant.

CONCLUSIONS

These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

CD4(+) T cell effector molecules, in particular TNF-alpha and CD154, activate endothelial cells. However, the relative contributions of TNF-alpha and CD154 in mediating endothelial cell activation during complex Ag-driven CD4(+) T cell-endothelial cell interactions are not known. We utilized an in vitro model of CD4(+) T cell-endothelial cell interactions to characterize the contributions of TNF-alpha and CD154 in mediating upregulation of adhesion molecules CD54, CD62E, and CD106 on human umbilical vein endothelial cells (HUVEC). HUVEC were first treated with IFN-gamma to upregulate MHC Class II expression. IFN-gamma minimally effects HUVEC adhesion molecule expression but renders them capable of MHC class II restricted interactions with CD4(+) T cells. Coculturing MHC class II+ HUVEC and CD4(+) T cells with the superantigen SEB induces a rapid and marked upregulation of CD54, CD62E, and CD106 expression on HUVEC, as shown by FACS analysis. To study the effector molecules mediating SEB-driven, CD4(+) T cell-dependent endothelial cell activation, similar experiments were performed in the presence of neutralizing anti-CD154, anti-TNF-alpha, or anti-IL1 antibodies, as well as combinations of these antibodies. In contrast to the anti-CD154 or anti-IL-1 antibodies, the anti-TNF-alpha mAb markedly inhibited SEB-dependent, CD4(+) T cell-induced HUVEC activation. We conclude that TNF-alpha, not CD154, plays the major role in SEB-driven, CD4(+) T cell-induced endothelial cell activation in vitro.

Fumaric acid esters, dimethyl fumarate (DMF) in particular, have been established for the therapy of psoriasis and, more recently, multiple sclerosis. In the light of therapy-limiting dose-dependent side effects, such as gastrointestinal irritation, reducing the effective doses of FAE is a worthwhile goal. In search of strategies to maintain the anti-inflammatory activity of DMF at reduced concentrations, we found that NF-κB inhibition augmented key anti-inflammatory effects of DMF in two complementary experimental settings in vitro. At non-toxic concentrations, both proteasome inhibition with bortezomib as well as blocking NF-κB activation through KINK-1, a small molecule inhibitor of IKKβ-profoundly enhanced DMF-dependent inhibition of nuclear NF-κB translocation in TNFα-stimulated human endothelial cells. This resulted in significant and selective co-operative down-regulation of endothelial adhesion molecules crucial for leucocyte extravasation, namely E-selectin (CD62E), VCAM-1 (CD106) and ICAM-1 (CD54), on both mRNA and protein levels. Functionally, these molecular changes led to synergistically decreased rolling and firm adhesion of human lymphocytes on TNF-activated endothelial cells, as demonstrated in a dynamic flow chamber system. If our in vitro findings can be translated into clinical settings, it is conceivable that anti-inflammatory effects of DMF can be achieved with lower doses than currently used, thus potentially reducing unwanted side effects.