Evonymus europaeus agglutinin as a marker of endothelial cells in the human

Vascular endothelial cells were marked with Evonymus europaeus agglutinin (EEA) in the major organs of 6 subjects to reveal the anatomical distribution of positive elements in the human after fixation in paraformaldehyde-lysine-periodate and embedding in paraffin. EEA may be used as a marker of human adult vascular endothelium in the same way as Ulex europaeus Agglutinin 1, revealing the same positive endothelial and epithelial territories. The intensity of labeling was constant in a given subject of the B blood group. In A or 0 individuals the cerebral endothelium showed a much lower reactivity than the cardiac endothelium. This lectin, equally active in higher mammals, offers the unique advantage of direct comparison between species.     

Production of B cell stimulatory factor-2/interleukin-6 activity by human endothelial cells

The effect of culture supernatants of endothelial cell (EC) lines on the immunoglobulin-M(IgM) synthesis by human B cell line, SKW6-CL4 cells, was investigated. Supernatants of human EC stimulated IgM synthesis, as high as 6-fold, but supernatants of bovine EC did not. This enhancing activity was completely blocked by addition of anti-human B cell stimulatory factor-2/interleukin-6 (BSF-2/IL-6) antibody. These data suggest that human EC might participate in the human antibody production system by producing soluble factor, BSF-2/IL-6.     

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.     

Soluble interleukin-2 receptor as a marker for progression of coronary artery calcification in type 1 diabetes

INTRODUCTION: Soluble interleukin-2 receptor (sIL2r), a marker of T cell activation, is elevated in inflammatory processes, such as rheumatoid arthritis, hepatitis and neoplasm. We explored a potential association between plasma sIL2r levels and progression of coronary artery calcification (CAC), a marker for subclinical atherosclerosis, in a prospectively followed cohort of type 1 diabetic and non-diabetic subjects, aged 20-59 years, with no history of coronary artery disease. MATERIALS AND METHODS: CAC progression was assessed by electron beam tomography over 2.6 years (range 1.6-3.2). Plasma sIL2r levels were measured in a nested case-control substudy of 98 subjects (67 diabetic, 31 non-diabetic) with and 173 subjects (84 diabetic, 89 non-diabetic) without significant CAC progression. Log-transformed sIL2r levels were analyzed by conditional logistic regression to compare subjects with and without significant CAC progression. RESULTS: SIL2r was a significant predictor for CAC progression after adjusting for presence of baseline CAC, age, gender, diabetes status, baseline calcium volume score and adiponectin (OR 1.99, 95% CI 1.09-3.61, p = 0.02 for a doubling of sIL2r level). Addition of BMI, LDL, HDL, hypertension, smoking status, HbA1c, CRP, fibrinogen, homocysteine and PAI-1 to regression models weakened but did not remove sIL2r as a predictor of CAC progression. There was no indication that this effect was different by diabetes status (p = 0.6 for diabetes-sIL2r interaction). DISCUSSION: Elevated plasma sIL2r is associated with CAC progression independent of traditional coronary artery disease risk factors in type 1 diabetic and non-diabetic young adults. SIL2r should be considered as a novel marker of inflammation leading to coronary artery disease.     

Calcyclin as a marker of human epithelial cells and fibroblasts

The distribution of calcyclin in human tissues was studied using polyclonal antibodies against this protein. In all organs examined (breast, heart, intestine, kidney, liver, ovary, placenta, stomach, thymus, and uterus) only epithelial cells and fibroblasts were stained. This suggests that calcyclin expression is related either to proliferation rate or secretion activity. The data show that calcyclin might be considered as a marker of some human epithelial cells and fibroblasts.     

Paracrine mitogenic effect of human endothelial progenitor cells: role of interleukin-8

Endothelial progenitor cells (EPCs) play an important role in repair of vascular injury and neovascularization. Molecular mechanisms underlying vascular effects of EPCs are not fully understood. The present study was designed to test the hypothesis that human EPCs exert a strong paracrine mitogenic effect on mature endothelial cells. Levels of interleukin-8 (IL-8) were significantly higher in conditioned medium (CM) collected from EPCs than in CM derived from mature endothelial cells [umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (CAECs)]. CM of EPCs stimulated proliferation of HUVECs and CAECs. This mitogenic effect was partially inhibited by IL-8-neutralizing antibody. In contrast, CM of HUVECs and CAECs had a weak or no mitogenic effect on mature endothelial cells. Our results demonstrate significantly higher levels of IL-8 secretion by human EPCs than by mature endothelial cells. IL-8 appears to be an important mediator of the paracrine mitogenic effect of EPCs.     

Gene expression pattern in human monocytes as a surrogate marker for systemic inflammatory response syndrome (SIRS).

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is a mild inflammatory episode which, in a minority of patients, may deteriorate into septic shock. In the mouse, injection of bacteria or bacterial endotoxin induces systemic inflammation through the activation of blood monocytes, which leads to lethal shock. A number of intervention strategies have been shown to prevent progression to shock in mouse model systems. However, recent clinical trials of a number of these therapeutic strategies in patients have been uniformly disappointing. In contrast to the situation in the mouse models, there may be many different ways to initiate systemic inflammation in patients and not all of them need necessarily involve activation of blood monocytes. If there is no unifying mechanism behind the induction of systemic inflammation in patients and no common rules governing its development, then it is unlikely that generally applicable therapeutic strategies will be found that can prevent progression into shock. MATERIALS AND METHODS: We used differential display to compare gene expression patterns in monocytes of recent-admission multi-trauma patients with clinically diagnosed SIRS to the patterns in monocytes of healthy controls. RESULTS: Of seven differentially displayed bands that were recovered and sequenced, five were associated with SIRS and two were preferentially expressed in the monocytes of healthy controls. CONCLUSION: The data show that monocytes of SIRS patients are in an activation state that is different from that of monocytes from the healthy controls, that monocytes from many individual patients share similar patterns of differentially expressed sequences, and that by this criterion, the multi-trauma SIRS patients are a remarkably coherent group.     

Serum amyloid A induces endothelial dysfunction in porcine coronary arteries and human coronary artery endothelial cells

The objective of this study was to determine the effects and mechanisms of serum amyloid A (SAA) on coronary endothelial function. Porcine coronary arteries and human coronary arterial endothelial cells (HCAECs) were treated with SAA (0, 1, 10, or 25 microg/ml). Vasomotor reactivity was studied using a myograph tension system. SAA significantly reduced endothelium-dependent vasorelaxation of porcine coronary arteries in response to bradykinin in a concentration-dependent manner. SAA significantly decreased endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein levels as well as NO bioavailability, whereas it increased ROS in both artery rings and HCAECs. In addition, the activities of internal antioxidant enzymes catalase and SOD were decreased in SAA-treated HCAECs. Bio-plex immunoassay analysis showed the activation of JNK, ERK2, and IkappaB-alpha after SAA treatment. Consequently, the antioxidants seleno-l-methionine and Mn(III) tetrakis-(4-benzoic acid)porphyrin and specific inhibitors for JNK and ERK1/2 effectively blocked the SAA-induced eNOS mRNA decrease and SAA-induced decrease in endothelium-dependent vasorelaxation in porcine coronary arteries. Thus, SAA at clinically relevant concentrations causes endothelial dysfunction in both porcine coronary arteries and HCAECs through molecular mechanisms involving eNOS downregulation, oxidative stress, and activation of JNK and ERK1/2 as well as NF-kappaB. These findings suggest that SAA may contribute to the progress of coronary artery disease.     

Testing for heterogeneity in the utility of a surrogate marker

In studies that require long-term and/or costly follow-up of participants to evaluate a treatment, there is often interest in identifying and using a surrogate marker to evaluate the treatment effect. While several statistical methods have been proposed to evaluate potential surrogate markers, available methods generally do not account for or address the potential for a surrogate to vary in utility or strength by patient characteristics. Previous work examining surrogate markers has indicated that there may be such heterogeneity, that is, that a surrogate marker may be useful (with respect to capturing the treatment effect on the primary outcome) for some subgroups, but not for others. This heterogeneity is important to understand, particularly if the surrogate is to be used in a future trial to replace the primary outcome. In this paper, we propose an approach and estimation procedures to measure the surrogate strength as a function of a baseline covariate W and thus examine potential heterogeneity in the utility of the surrogate marker with respect to W. Within a potential outcome framework, we quantify the surrogate strength/utility using the proportion of treatment effect on the primary outcome that is explained by the treatment effect on the surrogate. We propose testing procedures to test for evidence of heterogeneity, examine finite sample performance of these methods via simulation, and illustrate the methods using AIDS clinical trial data.     

Varicose veins as a source of adult human endothelial cells

Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.     

Epinephrine upregulates superoxide dismutase in human coronary artery endothelial cells

Regular exercise resulting in release of catecholamines is an oxidant stress, and yet it protects humans from acute cardiac events. We designed this study to examine the effect of epinephrine on free radical release and endogenous superoxide dismutase (SOD) gene and protein expression in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with epinephrine (10(-9) to 10(-5) M) alone or with the water-soluble analog of vitamin E (trolox) (10(-5) M), the lipid-soluble vitamin E (5 x 10(-5) M), or the beta(1)-adrenergic blocker atenolol (10(-5) M). At 1 and 24 h of incubation with epinephrine, superoxide anion generation increased by 102 and 81% in the HCAECs. There was a marked increase in both MnSOD and Cu/ZnSOD mRNA and protein, as determined by RT-PCR and Western Analysis, respectively. Both MnSOD and Cu/ZnSOD activities were also increased. Pretreatment of HCAECs with trolox and vitamin E decreased superoxide anion generation (p <.05 vs. epinephrine alone) and blocked the subsequent upregulation of SOD mRNA and protein. Treatment of cells with the beta-blocker atenolol also blocked the upregulation of SOD (p <.05 vs. epinephrine alone). These observations suggest that epinephrine via beta(1)-adrenoceptor activation causes superoxide anion generation, and the superoxide subsequently upregulates the endogenous antioxidant species SOD. These observations may be the basis of long-term benefits of exercise.