OBJECTIVE

Expression of endothelial leukocyte adhesion molecule (ELAM-1 or CD62E) plays a role as an early event of atherogenesis. It is well known that interleukin-1 (IL-1) expresses ELAM-1 on vascular endothelial cells. We have examined pathological factors that induce ELAM-1 expression on cultured endothelial cells.

METHODS

Examined factors were native low density lipoprotein (LDL), oxidized LDL, glycated LDL, hypoxia, and IL-1. Peroxidation of LDL was performed by ultraviolet radiation. Hypoxia was reproduced by adding a hypoxic cell-culture medium that was deoxygenated by use of a vacuum pump and nitrogen gas. Endothelial cells were harvested from a porcine aorta and were allowed to proliferate to be subconfluent in slide chambers. Expression of ELAM-1 was evaluated by counting the number of cells that were characterized by positive staining with the immunohistochemical technique.

RESULTS

Without any stimulants, about 6.9% of the endothelial cells expressed ELAM-1. Weakly oxidized LDL (12 pmol/micrograms protein) significantly expressed ELAM-1 (14.8%) after an incubation period of 1 hour. Glycated LDL induced significant expressions (12.6%) in a fructosamine concentration of 65 pmol/micrograms protein. A one-hour incubation with a hypoxic culture medium expressed ELAM-1 in 16.3% of the cells. Native LDL did not cause any significant increases in the percentage. IL-1 expressed ELAM-1 in 30% of the cells even with as low a concentration as 3.1 U/ml.

CONCLUSIONS

The present study shows that not only IL-1 but also weakly oxidized LDL, glycated LDL, and hypoxia may be possible factors that cause the expression of ELAM-1.

Staphylococcal scalded skin syndrome (SSSS) results from the effect of exfoliative-toxins produced by staphylococcal strains. The disease affects predominantly children, and is rare in adults. We report two cases of the adult type of SSSS. Corticotherapy, chronic alcohol abuse and epilepsy-related immune changes might have been predisposing factors in these patients. The immunopathological characteristics of the inflammatory cell infiltrate in adults SSSS have not been thoroughly explored so far in the literature. Biopsies from 2 patients with bullous SSSS skin were studied by means of immunochemistry using a panel of 10 antibodies directed to FXIIIa, CD15, CD31, CD45R0, CD50, CD54, CD62E, CD95, CD106, and L1-protein, respectively. Cutaneous biopsies from related blistering diseases were compared. They included drug-induced toxic epidermal necrolysis (TEN), bullous impetigo and superficial pemphigus. A dense cell infiltrate composed of granulocytes (CD15+), macrophages (L1 protein+) and memory T cells (CD45R0+) and a strong expression of ICAM-3 (CD50) were present in the epidermis. CD95+ keratinocytes were lining the intraepidermal blisters. Type I dermal dendrocytes (Factor XIIIa+) were numerous and plump in the dermis. Bullous impetigo exhibited the same pattern of inflammatory cells, but with a lower density in type I dermal dendrocytes. TEN differed from SSSS by both the absence of CD15+ granulocytes and a stronger expression of the pro-apoptotic CD95 antigen in the epidermis. In superficial pemphigus, CD95 antigen was not expressed, and CD15+ granulocytes, CD45R0+ lymphocytes and L1 protein+ monocytes were much less numerous. It is concluded that the specific binding of SSSS-induced exotoxins to the desmosomes alters the keratinocyte metabolism leading to an inflammatory reaction followed by focal apoptosis. Our findings are in line with the concept that SSSS exotoxins might be superantigens. A common pathomechanism leading to epidermal destruction is likely operative in SSSS and bullous impetigo. The inflammatory cell composition in TEN and superficial pemphigus markedly differs from that in SSSS.

OBJECTIVE

The objective of the study was to investigate the relationship of circulating endothelial-derived microparticls (EMP) pattern with body mass index (BMI) in CHF patients.

METHODS

The study retrospectively evolved 153 patients (86 males) who were underwent multispiral contrast-enhanced computed tomography angiography or conventional angiographic examination of coronary arteries. Flowcytometry analysis for quantifying the number of EMPs was used at baseline.

RESULTS

Using C-statistics for models with CHF, BMI, and circulating biomarkers (NT-pro-BNP, OPG and adiponectin) as continuous variables we found that adding of BMI to the based model (NYHA class of CHF) improved the relative IDI by 12.5% for increased CD31+/annexin V+ EMPs to CD62E+ EMPs ratio. When we used other model constructed on entering variables IDI appears to be improved up to 5.8% for increased EMPs (available for NT-pro-BNP as continuous variable). Three biomarkers (NYHA class of CHF+NT-pro-BNP+OPG) and four biomarkers (NYHA class of CHF+NT-pro-BNP+OPG+adiponectin) could not significantly improve predictive model based on combination of BMI and NYHA class of CHF for increased CD31+/annexin V+ EMPs to CD62E+ EMPs ratio.

CONCLUSIONS

We suggested that lower BMI is significant predictor for impaired phenotype of circulating EMPs in CHF patients.

OBJECTIVE

Several recent lines of evidence indicate that endothelial microparticles are a new biomarker that can be used to monitor endothelial dysfunction in coronary artery disease (CAD). However, data concerning the detection of small microparticles (diameter <0.5 µm) are lacking. The aim of this study was to detect small-size endothelial microparticles (SEMPs) in CAD patients to monitor endothelial dysfunction.

METHODS

In total, 19 CAD patients and 14 healthy subjects were recruited. The absolute numbers and percentages of CD31(+)/CD42b- SEMPs and CD62E(+) SEMPs were determined by flow cytometry. Clinical parameters were also recorded.

RESULTS

The mean percentage of CD62E(+) SEMPs was higher in the CAD patient group than in the healthy subject group. The area under the receiver operating characteristic curve of the percentage of CD62E(+) SEMPs was 0.795, and the cut-off value was 1.35. There was no correlation between the percentage of CD62E(+) SEMPs and various clinical parameters.

CONCLUSIONS

The percentage of CD62E(+) SEMPs is a potential biomarker for monitoring endothelial function in CAD.

BACKGROUND

Inheritance of Factor V Leiden (FVL) is associated with an increased but variable level of risk for thrombosis. We have previously shown that FVL heterozygotes have elevated levels of circulating pro-coagulant microparticles (MP). Here we sought to determine if these subjects differed in their plasma levels of FVL and if this was related to MP concentrations and/or history of thrombosis.

METHODS

The Hemoclot Quanti. V-L clotting assay was used to specifically measure FVL in plasma samples from 44 known carriers (12M, 32F; aged 46±13years). Circulating MP were quantified by flow cytometry using fluorochrome conjugated antibodies to platelet (CD41a), leukocyte (CD45), and endothelial (CD62e) surface markers, and MP prothrombinase activity was determined by ELISA.

RESULTS

The cohort was found to have a mean FVL of 49.5±5.6% and this was positively correlated to the total number of circulating CD41a+MP (R=0.31, p=0.03) but not to other MP subsets or to MP prothrombinase activity. The amount of FVL relative to normal factor V (FVL/FV clotting ratio) was calculated and found to be highly variable, ranging from 0.37 to 0.69, and significantly correlated with a history of thrombosis (n=14; p=0.04).

CONCLUSIONS

This is the first study to investigate the relationship between varying levels of FVL and plasma derived MP. These results are consistent with our previous findings of an increase in MP levels in carriers of FVL as compared to controls, and suggest a role for FVL/FV ratio in predicting risk of thrombosis in carriers of FVL.

BACKGROUND

Morbilliform rashes induced by amoxicillin are though to be caused by a delayed cell-mediated immune reaction. The importance of amoxicillin skin tests is not well defined. A better understanding of the mechanisms of amoxicillin-induced morbilliform rashes can be obtained by performing cutaneous immunohistological studies on specimens from amoxicillin-induced morbilliform rashes and positive amoxicillin skin test results.

METHODS

Skin biopsy specimens were obtained from 5 patients who had developed an amoxicillin-induced morbilliform rash. All patients underwent amoxicillin prick, patch, and intradermal tests. Similar immunohistological investigations were performed on amoxicillin-induced morbilliform rashes and positive skin test biopsy specimens, with a special focus on the expression of adhesion molecules. Three of the 5 patients developed delayed positive results to intradermal and patch tests and 2 patients developed delayed positive results to prick tests. Amoxicillin-induced morbilliform rashes were well reproduced by skin tests, with similar immunohistological results in amoxicillin-induced morbilliform rashes and skin test biopsy specimens. Keratinocytes were activated and expressed CD54 (intercellular adhesion molecule 1); perivascular lymphocytes were mostly CD2+, CD3+, and CD4+ and exhibited CD11a through CD18 (leukocyte function-associated antigen 1) and often HLA-DR and/or CD62L (leukocyte endothelial cell adhesion molecule 1); and endothelial cells were activated with a strong expression of CD54 (intercellular adhesion molecule 1), CD62E (endothelial leukocyte adhesion molecule 1), and CD31 (platelet endothelial cell adhesion molecule 1) in lesser amounts.

CONCLUSIONS

Findings of this clinical and immunohistochemical study support the theory of a T-cell-mediated immune reaction in patients with amoxicillin-induced morbilliform rashes, with a strong involvement of adhesion molecules both on endothelial and infiltrating cells. Our findings emphasize the importance of delayed readings of amoxicillin prick, intradermal, and patch tests.

Patients with bronchial asthma develop various types of asthmatic response to bronchial challenge with allergen, such as immediate/early asthmatic response (IAR), late asthmatic response (LAR) or delayed asthmatic response (DYAR), because of different immunologic mechanisms. The DYAR, occurring between 24 and 56 hours after the bronchial allergen challenge (p < 0.01), differs from IAR and LAR in clinical as well as immunologic features. This study investigates the expression of CD molecules (markers) on the surface of particular cell populations in the peripheral blood and their changes during the DYAR. In 17 patients developing the DYAR (p < 0.01), the bronchial challenge with allergen was repeated 2-6 weeks later. The repeated DYAR (p < 0.001) was combined with recording of CD molecule expression on various types of blood cells by means of flow cytometry up to 72 hours after the challenge. The results were expressed in percent of the mean relative fluorescence intensity. The DYAR was accompanied by (a) increased expression of CD11b, CD11b/18, CD16,CD32, CD35, CD62E, CD62L, CD64, and CD66b on neutrophils; CD203C on basophils; CD25 and CD62L on eosinophils; CD14, CD16, CD64, and CD86 on monocytes; CD3, CD4, CD8, CD11a, CD18, and CD69 on lymphocytes; CD16, CD56, CD57, and CD94 on natural killer (NK) cells; and CD31, CD41, CD61, CD62P, and CD63 on thrombocytes and (b) decreased expression of CD18 and CD62L on eosinophils, CD15 on neutrophils, and CD40 on lymphocytes. These results suggest involvement of cell-mediated hypersensitivity mechanism, on participation of Th1- lymphocytes, neutrophils, monocytes, NK cells, and thrombocytes in the DYAR.

BACKGROUND

Cell adhesion plays a pivotal role in most ocular surface inflammatory diseases. Adhesion molecules mediate cell-to-cell and cell-to-matrix adhesion. Their expression is up-regulated by pro-inflammatory stimuli such as cytokines, histamine or complement-derived anaphylatoxins. The dipeptide N acetyl-aspartyl glutamic acid (NAAGA) is used as unpreserved topical eyedrops in the treatment of allergic conjunctivitis. NAAGA is known to inhibit leukotriene synthesis, histamine release by mast cells, and complement-derived anaphylatoxin production.

OBJECTIVE

To investigate the potential capability of NAAGA to interfere with leukocyte adhesion to endothelial cells and modulate cytokine-induced expression of adhesion molecules.

METHODS

Human blood-derived leukocytes were co-cultured with human umbilical vein endothelial cells (HUVECs) in the absence or the presence of 1000 UI/mL human recombinant TNFalpha, 10(-4) M histamine di-hydrochloride or 5x10(-6) M human recombinant C5a, and in the absence or presence of NAAGA (final concentration 2.45%). Adhesion of leukocytes to HUVECs was calculated by subtracting the number of nonadherent leukocytes from the total number of leukocytes. Expression of adhesion molecules was assessed by flow cytometry using anti-CD11b, anti-CD49d, anti-ICAM-1 (CD54), anti-ICAM-2 (CD102), anti-VCAM-1 (CD106) and anti-ELAM-1 (CD62E) monoclonal antibodies.

RESULTS

NAAGA was found to totally inhibit adhesion of unstimulated leukocytes, or leukocytes activated with C5a, TNFalpha, or histamine, to TNFalpha-stimulated HUVECs (P=0.0001). Adhesion of leukocytes to unstimulated HUVECs was not modified by NAAGA. Similar results were obtained with endothelial cells stimulated by histamine or C5a. Taken together, these data indicate that NAAGA totally abrogates cell adhesion under inflammatory conditions, without interfering with the physiological adhesion of leukocytes to normal endothelium. At the molecular level, NAAGA inhibited histamine-induced expression of CD11b (P=0.0004) and CD49d (P=0.0045) on granulocytes. On TNFalpha-activated HUVECs, NAAGA induced a significant decrease in the VCAM-1 expression level (P<0.0001) and totally reversed TNFalpha-induced overexpression of ICAM-1 (P=0.0069), ICAM-2 and ELAM-1 (P<0.0001), without interfering with baseline expression of these molecules.

CONCLUSIONS

These results show that the antiallergic compound NAAGA directly inhibits leukocyte adhesion to endothelial cells induced by pro-inflammatory stimuli, and abrogates TNFalpha-induced expression of adhesion molecules on granulocytes and endothelial cells. This capacity to block overexpression of selectins and integrins induced by pro-inflammatory stimuli confers to NAAGA a potential as an anti-inflammatory drug, since interfering with adhesion molecule expression is probably one of the most efficient ways to curb leukocyte recruitment to inflammatory sites.

High-energy particle radiation could have a considerable impact on health during space missions. This study evaluated C57BL/6 mice on Day 40 after total-body 56Fe26+ irradiation at 0, 1, 2 and 3 gray (Gy). Radiation consistently increased thymus mass (one-way ANOVA: P < 0.005); spleen, liver and lung masses were similar among all groups. In the blood, there was no radiation effect on the white blood cell (WBC) count or major leukocyte types. However, the red blood cell count, hemoglobin, hematocrit and the CD8+ T cytotoxic (Tc) cell count and percentage all decreased, while both the CD4:CD8 (Th:Tc) cell ratio and spontaneous blastogenesis increased, in one or more irradiated groups compared with unirradiated controls (P < 0.05 vs 0 Gy). In contrast, splenic WBC, lymphocyte, B cell and T helper (Th) counts, %B cells and the CD4:CD8 ratio were all significantly elevated, while Tc percentages decreased, in one or more of the irradiated groups compared with controls (P < 0.05 vs 0 Gy). Although there were trends for minor, radiation-induced increases in %CD11b+ granulocytes in the spleen, cells double-labeled with adhesion markers (CD11b+CD54+, CD11b+CD62E+) were normal. Splenocyte spontaneous blastogenesis and that induced by mitogens (PHA, ConA, LPS) was equivalent to normal. In bone marrow, the percentage of cells expressing stem cell markers, Sca-1 and CD34/Sca-1, were low in one or more of the irradiated groups (P < 0.05 vs 0 Gy). Collectively, the data indicate that significant immunological abnormalities still exist more than a month after 56Fe irradiation and that there are differences dependent upon body compartment.

BACKGROUND

The experimental use of cultured endothelial cells derived from the microvasculature such as glomerular endothelial cells possesses many problems, including limited growth rates, heterogeneity and loss of specific cell properties dependent on culture passage. In this study, we attempted to establish immortalized, human glomerular endothelial cell (HGEC) lines.

METHODS

HGECs of up to 5 passages were transformed by infection with simian virus (SV)-40. After 4-6 weeks the surviving, foci-forming cells were harvested and cloned. Each cell line obtained was examined by immunofluorescence with antibodies to antigens specific for vascular endothelial cells. The expression of adhesion molecules on cells incubated with or without TNF-alpha was also examined by cellular ELISA.

RESULTS

Three of twelve cell lines obtained expressed SV40 large T-antigen and von Willebrand's factor, as well as endothelial cell adhesion molecules including ICAM-1 (CD54), PECAM-1 (CD31) and E-selectin (CD62E). In these cells, ICAM-1 and E-selectin expression was up-regulated by TNF-alpha, as in native cultured HGEC.

CONCLUSIONS

These cell lines maintain the morphologic and functional characteristics of HGEC even after 60 passages. Immortalized HGEC will be useful for research on glomerular cell biology and provide a standardized substrate for anti-endothelial cell antibody detection.

BACKGROUND

Washing of red blood cells (RBC) can reduce unwanted biological response modifiers (BRMs) that can mediate transfusion complications in infants. The aim of this study was to examine the in vitro quality and the changes in BRMs following washing in paediatric RBC units.

METHODS

A pool and split design was used to prepare RBC (either 1 or 4 days old; n = 26 pairs). One unit was washed with 0·9% saline by centrifugation and then resuspended in SAG-M, while the other remained unwashed. Each RBC unit was divided to produce four units of paediatric-sized components. Samples were taken after 3 h and subsequently on days 1, 2, 7 and 14 post-wash.

RESULTS

Washing of RBC resulted in some red cell loss, with a minor increase in haemolysis. Washing effectively reduced supernatant potassium and IgA, as well as cytokines and complement proteins. RBC microparticles were significantly reduced in RBC washed at 1, but not 4 days post-collection. Incubation with supernatant from unwashed but not washed RBC led to endothelial cell activation, with increased cell surface expression of CD62E (E-selectin) and CD106 (VCAM).

CONCLUSIONS

Although washing affected some aspects of the in vitro quality of RBC, it effectively reduced the concentration and activity of BRMs in the supernatant of RBC. Such a reduction may be clinically beneficial in selected patient groups.

OBJECTIVE

To develop a sensitive and reproducible technique for measuring the adherence of blood lymphocytes to vessel walls exposed in sections of human retina and for examining the role of lymphocyte and vascular adhesion molecules in these events.

METHODS

Cryostat sections of human retina were overlaid with blood lymphocytes from healthy subjects, and experimental conditions were sought by which preferential attachment of the cells occurred to blood vessel walls in the retinal sections. Adherent lymphocytes were identified by staining with methyl green-thionine, and transected blood vessels were identified by their structure and by staining of basement membranes with periodic acid-Schiff. The adherence of enriched preparations of CD4+ (T-helper) and CD8+ (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Behçet's disease was examined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukocyte integrins to lymphocyte binding was studied by blocking experiments with monoclonal antibodies.

RESULTS

The optimal selectivity of blood lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf serum and overlaid onto retinal sections for 30 minutes at 23 degrees C with gentle agitation. Under these conditions, 92% of the lymphocytes that adhered to the section were confined to the retinal microvasculature, and CD4+ T cells were more adherent than CD8+ T cells (P < 0.01). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Behçet's disease showed supranormal vascular adherence (P < 0.005). Many transected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICAM-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion molecules occupied < 20% of the area of the blood vessel walls. Lymphocyte adhesion to the retinal vessels was more dependent on CD29 (the common chain of the beta 1 integrins) expression than either CD11a/CD18 or CD49d.

CONCLUSIONS

This technique allows measurements to be made of lymphocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence to sections of pathologic retina may be of clinical and experimental applicability in understanding mechanisms of retinal inflammation.

Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.

BACKGROUND

Inflammatory genes may be unevenly expressed in different heart chambers.

METHODS

Biopsies were taken simultaneously from the right atrium (RA), left atrium (LA), and left ventricle (LV) of 19 patients before cardioplegic arrest during open heart surgery. The mRNA expression of tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1 (ET-1), E-selectin (CD62E), intercellular adhesion molecule-1 (ICAM-1) and its ligand CD18, and CD25 was evaluated with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

Expression of TNFalpha mRNA was higher in RA than LA and LV (p<0.05), whereas IL-1beta was more expressed in LA than RA (p<0.05), which was higher than LV (p<0.0001). There were no significant regional differences in the expression of ICAM-1, CD62E, CD25, iNOS, and eNOS. CD18 was higher in RA than LA (p<0.05); ET-1 was more expressed in RA than LV (p<0.04). Patients with stable angina had no expression of eNOS.

CONCLUSIONS

Gene expression of inflammatory mediators was detected in the hearts of patients with different cardiovascular disorders, and was unevenly distributed in different heart chambers. Cardiac biopsies should be taken from the same site.

In this manuscript we describe a potentially new mechanism by which unstimulated human monocytes activate endothelial cells (EC) through the secondary induction of endothelial tumour necrosis factor alpha (TNF-alpha). Serum free supernatants (SN) of peripheral blood mononuclear cells (PBMC) strongly induce the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), vascular cell adhesion molecule 1 (VCAM-1, CD106), and endothelial-leukocyte adhesion molecule 1 (ELAM-1, CD62E) on human EC 24 and 4 h post treatment, respectively. Further characterization of the responsible subpopulation revealed the CD14+ monocytes and a monocytic cell line (MM6) to produce an endothelial activating factor (EAF). The EAF also triggers an adhesion and a transendothelial migration (TEM) of peripheral blood cells. Using neutralization with an anti TNF-alpha MoAb MAK195, EAF is not identical with TNF-alpha, but induces the expression of endothelial TNF-alpha, since MAK195 blocked TEM only when coincubated with EC, not with monocytes. Furthermore, intracellular TNF-alpha was significantly upregulated in EC after treatment with SN-MM6. Another evidence for a secondary autocrine mechanism was provided by culturing the EC with a conditioned medium of SN-MM6 treated EC. This conditioned medium induces an adhesion molecule expression and TEM in a similar way to SN-MM6 and can completely be inactivated by anti TNF-alpha. Taken together, these data may have an impact for, e.g. transplantational settings that donor monocytes may trigger an inflammatory response in the absence of further activation signals by eliciting an endogenous TNF-alpha response in the host.

BACKGROUND

The interaction between host lymphocytes and graft endothelial cells plays an important role in graft rejection.

METHODS

Using our model of isolated ventilated lung from female mouse perfused with fresh blood from either isogeneic or allogeneic male mouse for 3 hours without noticeable ischemia, we have investigated the kinetics of the early events after endothelial cell triggering by E-selectin engagement.

RESULTS

Isogeneic perfusion induced nonspecific endothelial cell activation, which was characterized by up-regulation of E-selectin, intercellular adhesion molecule (ICAM)-1, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and lymphotoxin-alpha (mRNAs by real-time polymerase chain reaction). Allogeneic perfusion was characterized after 3 hours by an additional loose adhesion of lymphocytes mediated by the E-selectin and related to the allogeneic activation of endothelial cells. These in turn expressed the I-A molecule (immunostaining). ICAM-1 and lymphocyte function-associated antigen (LFA)-3 mRNA levels were significantly increased in lung extracts after 2 hours, then vascular cell adhesion molecule (VCAM)-1 and TNF-alpha mRNAs after 3 hours without evidence of TNF-alpha production (enzyme-linked immunoadsorbent assay). The major participation of the E-selectin in early allogeneic activation by way of the protein kinase (PK)C pathway was confirmed by using a neutralizing anti-CD62E monoclonal antibody or the inhibitory PKC 19-31 fragment.

CONCLUSIONS

Altogether, our results demonstrate that E-selectin expression (1) is not a consequence of TNF-alpha triggering, (2) up-regulates its own expression and expression of I-A, VCAM-1, TNF-alpha, and lymphotoxin-alpha mRNAs, and (3) down-regulates expression of LFA-3 and ICAM-1 mRNAs. In conclusion, in our physiologic model, the E-selectin highly participates in the loose adhesion of allogeneic lymphocytes and in the early activation of endothelial cell and therefore in structural and functional lung alterations.

BACKGROUND

The prevalence of food allergies and other allergic reactions is increasing worldwide, particularly in highly-urbanized populations. Cell adhesion molecules are expressed in response to various pro-inflammatory cytokines. The expression of intercellular adhesion molecule 1 - ICAM-1 (CD54), ICAM-1 (CD106), P-selectin (CD62P), and E-selectin (CD62E) on vascular endothelial cells is induced by such pro-inflammatory cytokines as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1).

OBJECTIVE

To analyze concentrations of E-selectin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in patients with an allergic type of food sensitivity co-existing with gastritis and to compare them to the values determined in individuals with dyspeptic symptoms not associated with allergic disorders.

METHODS

The study included 80 patients, among them 50 individuals with food sensitivity confirmed based on compulsory standards, and 30 subjects with dyspeptic symptoms not accompanied by allergic conditions. Venous blood samples were taken from each patient and concentrations of E-selectin and PECAM-1 were determined by means of ELISA.

RESULTS

Mean concentrations of sE-selectin and sPECAM-1 in patients with food allergy amounted to 54.0 ±21.6 ng/ml and 132.8 ±31.4 ng/ml, respectively. In individuals without food allergy, mean concentrations of sE-selectin and sPECAM-1 were 57.7 ±17.9 ng/ml and 139.6 ±31.1 ng/ml, respectively. Patients with food allergy and individuals with dyspeptic symptoms not associated with food allergy did not differ significantly in terms of sE-selectin concentrations (Mann-Whitney U-test, p = 0.453028). Similarly, no significant intergroup differences were observed with regard to sPECAM-1 concentrations (Mann-Whitney U-test, p = 0.231054).

CONCLUSIONS

Adhesion molecules play an important role in the development of inflammation. This study did not find significant differences in the concentrations of such molecules as sE-selectin and sPECAM-1 between patients with food allergy and gastritis, and subjects in whom gastritis was not accompanied by atopic disorders. A positive correlation between the concentrations of sPECAM-1 and E-selectin was observed in food allergy patients. Consequently, it can be concluded that these molecules participate in the pathogenesis of the inflammatory process independently of the etiopathogenesis of gastritis.

In order to identify the factors that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 x 10(6) lymphocytes were overlaid onto brain sections for 30 min at 7 degrees C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d (p < or = 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.

Background Acute vascular effects of high intensity physical activity are incompletely characterized. Circulating microparticles are cellular markers for vascular activation and damage. Methods Microparticles were analysed in 99 marathon runners (49 ± 6 years, 22% female) of the prospective Berlin Beat of Running study. Blood samples were taken within three days before, immediately after and within two days after the marathon run. Endothelial-derived microparticles were labelled with CD144, CD31 and CD62E, platelet-derived microparticles with CD62P and CD42b, leukocyte-derived microparticles with CD45 and monocyte-derived microparticles with CD14. Results Marathon running induced leukocytosis (5.9 ± 0.1 to 14.8 ± 0.3 109/l, p < 0.0001) and increased platelet counts (239 ± 4.6 to 281 ± 5.9 109/l, p < 0.0001) immediately after the marathon. Blood monocytes increased and lymphocytes decreased after the run ( p < 0.0001). Endothelial-derived microparticles were acutely increased ( p = 0.008) due to a 23% increase of apoptotic endothelial-derived microparticles ( p = 0.007) and returned to baseline within two days after the marathon. Thrombocyte-derived microparticles acutely increased by 38% accompanied by an increase in activated and apoptotic thrombocyte-derived microparticles ( p ≤ 0.0001) each. Both monocyte- and leukocyte-derived microparticles were decreased immediately after marathon run ( p < 0.0001) and remained below baseline until day 2. Troponin T increased from 12 to 32 ng/l ( p < 0.0001) immediately after the run and returned to baseline after two days. Conclusion Circulating apoptotic endothelial- and thrombocyte-derived microparticles increased after marathon running consistent with an acute pro-thrombotic and pro-inflammatory state. Exercise-induced vascular damage reflected by microparticles could indicate potential mechanisms of post-exertional cardiovascular complications. Further studies are warranted to investigate microparticles as markers to identify individuals prone to such complications.

What is the central question of this study? What is the effect of exercise intensity on circulating microparticle populations in young, healthy men and women? What is the main finding and its importance? Acute, moderate-intensity continuous exercise and high-intensity interval exercise altered distinct microparticle populations during and after exercise in addition to a sex-specific response in CD62E+ microparticles. The microparticles studied contribute to cardiovascular disease progression, regulate vascular function and facilitate new blood vessel formation. Thus, characterizing the impact of intensity on exercise-induced microparticle responses advances our understanding of potential mechanisms underlying the beneficial vascular adaptations to exercise.

Circulating microparticles (MPs) are biological vectors of information within the cardiovascular system that elicit both deleterious and beneficial effects on the vasculature. Acute exercise has been shown to alter MP concentrations, probably through a shear stress-dependent mechanism, but evidence is limited. Therefore, we investigated the effect of exercise intensity on plasma levels of CD34+ and CD62E+ MPs in young, healthy men and women. Blood samples were collected before, during and after two energy-matched bouts of acute treadmill exercise: interval exercise (10 × 1 min intervals at ∼95% of maximal oxygen uptake V̇O2max) and continuous exercise (65% V̇O2max). Continuous exercise, but not interval exercise, reduced CD62E+ MP concentrations in men and women by 18% immediately after exercise (from 914.5 ± 589.6 to 754.4 ± 390.5 MPs μl-1 ; P < 0.05), suggesting that mechanisms underlying exercise-induced CD62E+ MP dynamics are intensity dependent. Furthermore, continuous exercise reduced CD62E+ MPs in women by 19% (from 1030.6 ± 688.1 to 829.9 ± 435.4 MPs μl-1 ; P < 0.05), but not in men. Although interval exercise did not alter CD62E+ MPs per se, the concentrations after interval exercise were higher than those observed after continuous exercise (P < 0.05). Conversely, CD34+ MPs did not fluctuate in response to short-duration acute continuous or interval exercise in men or women. Our results suggest that exercise-induced MP alterations are intensity dependent and sex specific and impact MP populations differentially.

UNASSIGNED

Bariatric surgery is a widely adopted treatment for obesity and its secondary complications. In the past decade, microvesicles (MVs) and CD36 have increasingly been considered as possible biomarkers for obesity, the metabolic syndrome (MetSy), type 2 diabetes mellitus (T2DM). Thus, the purpose of this study was to investigate how weight loss resulting from bariatric surgery affects levels of specific MV phenotypes and their expression of CD36 scavenger receptor. Additionally, we hypothesised that subjects with MetSy had higher baseline concentrations of investigated MV phenotypes.

UNASSIGNED

Twenty individuals undergoing Roux-en-Y gastric bypass surgery were evaluated before and 3 months after surgery. MVs were characterised by flow cytometry at both time points and defined as lactadherin-binding particles within a 100-1000 nm size gate. MVs of monocyte (CD14) and endothelial (CD62E) origin were defined by cell-specific markers, and their expression of CD36 was investigated.

UNASSIGNED

Following bariatric surgery, subjects incurred an average BMI reduction (delta) of - 8.4 ± 1.4 (p < 0.0001). Significant reductions were observed for the total MVs (- 66.55%, p = 0.0017) and MVs of monocyte (- 36.11%, p = 0.0056) and endothelial (- 40.10%, p = 0.0007) origins. Although the bulk of CD36-bearing MVs were unaltered, significant reductions were observed for CD36-bearing MVs of monocyte (- 60.04%, p = 0.0192) and endothelial (- 54.93%, p = 0.04) origin. No differences in levels of MVs were identified between subjects who presented with MetSy at baseline (n = 13) and those that did not (n = 7).

UNASSIGNED

Bariatric surgery resulted in significantly altered levels of CD36-bearing MVs of monocyte and endothelial origin. This likely reflects improvements in ectopic fat distribution, plasma lipid profile, low-grade inflammation, and oxidative stress following weight loss. Conversely, however, the presence of MetSy at baseline had no impact on MV phenotypes.

What is the central question of this study? Are there sex-related differences in the number of circulating endothelial microparticles (EMPs) and microparticle microRNA expression in middle-aged adult humans? What is the main finding and its importance? Although the numbers of circulating endothelial microparticles do not differ between middle-aged men and women, there are sex-related differences in the expression of miR-125a in activation-derived EMPs and miR-34a in apoptosis-derived EMPs. Differences in circulating endothelial microparticle microRNA content may provide new insight into the sex-related disparity in the risk and prevalence of vascular disease in middle-aged adults. The aims of this study were to determine: (i) whether circulating concentrations of endothelial microparticles (EMPs) differ in middle-aged men compared with women; and (ii) whether there are sex-related differences in microRNA expression in EMPs. Peripheral blood was collected from 30 sedentary adults: 15 men (56 ± 6 years old) and 15 women (56 ± 5 years old). Endothelial microparticles were defined by markers of activation (CD62e+ ) or apoptosis (CD31+ /CD42b- ) by flow cytometry. Expression of microRNA (miR-34a, 92a, 125a and 126) in activation- and apoptosis-derived EMPs was measured by RT-PCR. Circulating activation- (33 ± 31 versus 39 ± 35 microparticles μl-1 ) and apoptosis-derived EMPs (49 ± 54 versus 42 ± 43 microparticles μl-1 ) were not significantly different between men and women. Expression of miR-125a (2.23 ± 2.01 versus 6.95 ± 3.99 a.u.) was lower (∼215%; P < 0.05) in activation-derived EMPs, whereas expression of miR-34a (1.17 ± 1.43 versus 0.38 ± 0.35 a.u.) was higher (∼210%; P < 0.05) in apoptosis-derived EMPs from men compared with women. Expression of microRNA in circulating EMPs may provide new insight into sex-related differences in cardiovascular disease.