Inflammation plays a critical role in the pathogenesis of atherothrombosis. Our aim was to examine the association between plasma concentrations of inflammatory biomarkers and severity and outcome of acute brain ischaemia. Plasma samples were collected within 36 h of symptom onset in patients with acute brain ischaemia, and assessed by conventional ELISA kits for concentration of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1). Patients were assessed serially for stroke severity (National Institute of Health stroke scale) and outcome during follow-up (modified Rankin Scale, mRS; and Stroke Impact Scale-16, SIS). Patients (n = 113, 65% men, mean age 64 +/- 12 years) had a mean IL-6 concentrations of 5.1 +/- 5.0 pg/ml and sICAM-1 of 377 +/- 145 ng/ml. IL-6, but not sICAM-1, concentrations were strongly associated with stroke severity (P < 0.01 at all serial assessments). Ln-transformed IL-6 levels (per 1 SD) were associated with disability (mRS > or = 2, OR = 1.7; 95% CI 1.1-3.0) and poor physical function (SIS < or = 85, OR = 1.7; 95% CI 1.0-2.8). Further adjustment for baseline stroke severity, however, eliminated these associations. Our results suggest that high plasma concentrations of the inflammatory biomarker IL-6 but not sICAM-1 are associated with stroke severity and poorer functional outcome. IL-6 does not add, however, additional prognostic information for stroke outcome beyond that conveyed by the stroke severity.

The antiphospholipid (APLA) syndrome is defined as the occurrence of venous or arterial thromboembolism or recurrent fetal loss in patients with a positive anticardiolipin (aCL) or lupus anticoagulant test on two occasions, 3 months apart. In this study, we sought to determine whether the level of C-reactive protein (CRP), a non-specific inflammatory marker, at the time of the initial positive aCL test is a predictor of the transient vs. sustained nature of the aCL. Patients with a positive aCL antibody test underwent high sensitivity (hs) CRP testing. A repeat aCL antibody assay was performed on a serum sample obtained 3-4 months later. The positive and negative predictive values of the hs-CRP assay as a predictor of the second aCL result were calculated and correlation between the hs-CRP result and second aCL result was determined. Fifty-four consecutive patients with a positive aCL test were studied. Forty-two had a positive aCL-2. The predictive value of a negative CRP test for a positive aCL-2 was found to be 82.5% (33 of 42 patients). A positive CRP was able to predict a negative aCL-2 in 35.7% of cases (nine of 14 patients). Our results suggest that the hs-CRP test may be useful in the assessment of patients with a positive aCL antibody test. A concurrently performed negative hs-CRP test result may be useful in making diagnostic and therapeutic decisions.

Background: CD40 ligand (CD40L) is a trimeric, transmembrane protein of the tumor necrosis factor family and together with its receptor CD40 is an important contributor to the inflammatory processes that lead to atherosclerosis, plaque destabilization, and thrombosis.Methods: In order to assess the association between serum concentrations of CD40 ligand (CD40L) and risk of future ischemic stroke and coronary events among patients with chronic CHD, we obtained baseline serum samples from patients (n = 3090) with chronic CHD enrolled in a secondary prevention trial. With a prospective nested case-control design, we measured baseline CD40L concentration in sera of patients who subsequently developed myocardial infarction, sudden cardiac death or ischemic stroke during follow-up (cases, n = 233) and in 233 age- and gender-matched pairs without any subsequent cardiovascular events.Results: Relative odds for recurrent cardiovascular events per one natural log unit difference of CD40L were 0.97 (95%Cl, 0.82-1.16). No increase in relative odds for recurrent cardiovascular events was observed per increasing quartiles of CD40L concentrations. In analysis for individual end-points, different trends of risks were observed beyond the 95 percentile for ischemic stroke (OR 2.22; 95%Cl, 0.46-12.5) and for recurrent coronary events (OR 0.35; 95%CI, 0.07-1.37), but falling short of statistical significance.Conclusion: High serum concentrations of CD40L were not associated with increased risk of ischemic stroke or coronary events in patients with chronic coronary heart disease. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

BACKGROUND:Monocyte chemoattractant protein-1 (MCP-1) participates in the recruitment of mononuclear cells to the vessel wall. HYPOTHESIS:The aim of the study was to evaluate the potential association between serum concentration of MCP-1 and risk of future cardiovascular events in patients with chronic coronary heart disease. METHODS:A nested case control design was used. Baseline serum samples were taken from patients with coronary heart disease who were enrolled in a secondary prevention study. The MCP-1 levels were measured in those patients who had recurrent cardiovascular events during follow-up (n = 233) and compared with levels in age- and gender-matched controls. RESULTS:There were no differences in serum MCP-1 levels between cases and controls. The relative odds of a recurrent cardiovascular event for each 1 standard deviation difference in MCP-1 serum concentration (160 pg/ml) was 1.19 (95% confidence interval, 0.95-1.45). No increase in the relative odds for recurrent cardiovascular events was observed per increasing tertiles of MCP-1 concentrations. CONCLUSION:Elevated MCP-1 levels are not associated with long-term risk of cardiovascular events in patients with stable coronary disease.

Background: Thrombophilia refers to series of acquired and inherited conditions that confer a tendency to thrombus formation. The exact relationship between thrombophilia and MI is not well established. Objectives: To determine the prevalence of thrombophilia, in young patients with their first MI and few conventional risk factors. Methods: We evaluated the baseline characteristics and the thrombophilia profile, including anti-cardiolipin antibodies, activated protein C resistance (APCR) with the factor V Leiden mutation, prothrombin G20210A mutation, protein C, protein S, and antithrombin-III levels, among 85 consecutive patients (< 50 year old) who were admitted to CCU with their first MI. Patients were divided into two groups: group A-patients with <= 1 risk factor and group B-patients with >= 2 risk factors. Results: 92% were male and 55% with anterior wall MI. Overall, the risk factor profile was: smoking in 60%, hyperlipidemia in 42%, positive family history in 29%, hypertension in 18%, diabetes mellitus in 13%, and obesity in 8%. Forty-seven percent of patients had <= 1 risk factor (n = 40, group A) and 53% had >= 2 risk factors (it = 45, group 13). The prevalence of the prothrombin mutation was 15% in group A compared to 7% in group B (p = 0.12). APCR secondary to a heterozygous genotype of factor V Leiden mutation was found in 20% in group A compared to 2% in group B (p < 0.01). Anti-cardiolipin antibodies were found in 16% in group A compared to 22% in group B (p = ns). Finally, we have found that the likelihood of identifying at least one thrombophilia marker was 50% in group A compared to 29% in group B (p = 0.046). Conclusions: The likelihood to detect at least one thrombophilia marker in young patients with MI and few conventional risk factors is significantly high. Thrombophilia may contribute to the development of MI in this specific group of young patients. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Aim: To evaluate the association between previous exposure to Chlamydia pneumoniae and future coronary risk in patients with coronary heart disease. Methods: A prospective, nested, case-control design was used. The patient sample was derived from a trial study of bezafibrate for the treatment of coronary heart disease. Anti-Chlamydia pneumoniae antibodies (IgG and IgA) in the baseline sera of 136 patients who had coronary events during follow-up (mean 6.2 years) were compared with those in 136 age- and gender-matched patients from the same trial without subsequent coronary events. Results: Mean titers of IgG and IgA antibodies were similar in cases and controls. The relative odds of future coronary events in patients who were seropositive at baseline were 1.0 (95% CI, 0.54–1.84) for IgG and 0.74 (95% CI, 0.41–1.31) for IgA. The relative odds did not change after adjustment for multiple confounding variables. The risk of future coronary events did not increase with increasing anti-Chlamydia pneumoniae antibody titers. Conclusions: Prior exposure to Chlamydia pneumoniae in patients with chronic coronary heart disease is not associated with increased risk of recurrent coronary events.

BACKGROUND AND PURPOSE:Inflammation is considered to be involved in the pathogenesis of ischemic stroke. Our purpose was to assess the role of soluble intercellular adhesion molecule-1 (sICAM-1) concentration, a marker of inflammation, in predicting future ischemic stroke among patients at risk because of chronic coronary heart disease. METHODS:We obtained baseline serum samples from patients with chronic coronary heart disease enrolled in the Bezafibrate Infarction Prevention trial (n=3090), which assessed the efficacy of bezafibrate in secondary prevention. Using a prospective nested case-control design, we measured baseline sICAM-1 concentration in sera of patients who developed ischemic stroke during a mean follow-up of 8.2 years (cases, n=134) and in age- and sex-matched controls without any subsequent cardiovascular events (n=134). RESULTS:Baseline serum concentrations of sICAM-1 were significantly higher in cases compared with controls (379 versus 350 ng/mL, P<0.05). sICAM-1 concentration at the highest quartile (>394 ng/mL) was associated with significantly higher relative odds of ischemic stroke compared with the lower concentrations after adjustment for potential confounding variables (relative odds, 2.1; 95% CI, 1.1 to 4.3). After fibrinogen and total white blood cell count were added to the multivariable model, the relative odds were 2.1 (95% CI, 1.1 to 4.2) and 2.2 (95% CI, 1.1 to 4.8), respectively. The risk associated with raised concentrations of sICAM-1 seemed to be highest for large disabling strokes of cardioembolic origin. CONCLUSIONS:Elevated concentrations of sICAM-1, a marker of inflammation, are associated with increased risk of ischemic stroke, independent of other traditional cerebrovascular risk factors and of plasma fibrinogen, among patients at increased risk because of manifest coronary heart disease.

OBJECTIVES:The goal of this study was to assess soluble intercellular adhesion molecule-1 (sICAM-1) level as a predictor of future acute coronary events in patients with chronic coronary heart disease (CHD). BACKGROUND:Increased sICAM-1 concentration has been shown to be associated with the incidence of CHD in healthy persons. Its significance in patients with CHD has been scarcely investigated. METHODS:We designed a prospective, nested case-control study. Sera were collected from patients with CHD enrolled in a secondary prevention trial that evaluated the efficacy of bezafibrate in reducing coronary events. We measured baseline sICAM-1 concentration in the sera of patients who developed subsequent cardiovascular events (cases: n = 136) during follow-up (mean: 6.2 years) and in age- and gender-matched controls (without events: n = 136). RESULTS:Baseline serum concentrations of sICAM-1 were significantly higher in cases versus controls (375 vs. 350 ng/ml; p < 0.05). Each 100 ng/ml increase in sICAM-1 concentration was associated with 1.27 (95% confidence interval [CI]: 1.00 to 1.63) higher relative odds of coronary events. Soluble ICAM-1 concentration in the highest quartile (>394 ng/ml) was associated with significantly higher odds of coronary events (compared with the lowest quartile), even after multivariate adjustment (2.31, 95% CI: 1.02 to 5.50). After adding fibrinogen and total white blood cell count to the multivariate model, the relative odds were 2.12 (95% CI: 0.88 to 5.35) and 2.70 (95% CI: 1.10 to 7.05), respectively. CONCLUSIONS:Elevated sICAM-1 concentration in CHD patients is associated with increased risk of future coronary events independent of other traditional risk factors.

Cyclophosphamide (CY), an alkylating cytostatic drug, is known for its ability to accelerate a number of experimental autoimmune diseases including spontaneous diabetes in NOD mice. The mechanism(s) by which CY renders autoreactive lymphocytes more pathogenic is largely unknown, but it has been postulated that the drug preferentially depletes regulatory (suppressor) T cells. It has been suggested that in cell-mediated autoimmune diseases, Th2-like lymphocytes secreting IL-4 and/or IL-10 provide protection, while Th1-like cells secreting IFN-gamma are pathogenic. In this study, we analysed the effects of CY on autoimmune diabetes and cytokines in two mouse models: the spontaneous diabetes of NOD mice and the diabetes induced in C57BL/KsJ mice by multiple injections of low dose streptozotocin (LD-STZ). In both models, CY induced severe lymphopenia and accelerated the progression to hyperglycemia. This was associated with changes in splenic cytokine patterns indicating a shift towards the IFN-gamma-secreting phenotype. We provide here evidence that IFN-gamma producers are relatively resistant to depletion by CY and that Th0 clones can be shifted towards Th1. However, direct exposure of T lymphocytes to CY may not be a necessary condition for exacerbation of diabetes; NOD.scid mice treated with CY before adoptive transfer of NOD splenocytes developed diabetes at a higher rate than did controls. Thus, the acceleration of diabetes by CY seems to be a complex event, which includes the relatively high resistance of IFN-gamma producers to the drug, their rapid reconstitution, and a Th1 shift of surviving T cell clones.

Until recently, the majority of patients with chronic urticaria (CU) have been determined to be idiopathic. Despite the absence of an obvious etiology, it is widely accepted that mast cell degranulation followed by perivascular cellular infiltration underlies the characteristic manifestations of this condition. Recently, there have been several reports of an increased frequency of thyroid autoimmunity in patients with CU.1,2 It has been suggested that the affected patients may have an underlying autoimmune process directed both at thyroid antigens and at antigens involved in mast cell degranulation.

T cells involved in autoimmune diseases have been characterized by the genetic elements used to construct their autoimmune TCR. In the present study, we sequenced the alpha and beta chains of the TCR expressed by a CD4(+) T cell clone, C9, functional in NOD mouse diabetes. Clone C9 can adoptively transfer diabetes or, when attenuated, C9 can be used to vaccinate NOD mice against diabetes. Clone C9 recognizes a peptide epitope (p277) of the 60 kDa heat shock protein (hsp60) molecule. We now report that the C9 TCR beta chain features a CDR3 peptide sequence that is prevalent among NOD mice. This CDR3 element is detectable by 2 weeks of age in the thymus, and later in the spleen and in the autoimmune insulitis. Thus, a TCR CDR3beta sequence appears to be a common idiotope associated with mouse diabetes.

Sarcoidosis (SA) and diffuse interstitial fibrosis (DIF) are characterized by alveolitis, mast cell hyperplasia and increased fibroblast proliferation. Stem cell factor (SCF) stimulates proliferation of hematopoietic progenitor cells involved in mast and stromal cell interaction. We assessed the role of SCF secreted by alveolar fibroblasts (AFb) in the development of fibrosis of DIF and SA in six patients with SA and six patients with DIF. Bronchoalveolar lavage (BAL) was performed by conventional methods. A total of 500 cells were differentially counted from Giemsa-stained cytopreps. AFb and supernatants were recovered from long-term cultures of BAL cells and from 24 h cultures of confluent AFb. Levels of SCF were measured by ELISA. Alpha actin content of AFb was characterized by immunohistochemistry. The expression of AFb mRNA for IL1-alpha and beta, TGF-beta, IFN-gamma, IL-2, IL-4, IL-5 and IL-6 was determined by RT-PCR. There was a lymphocytic predominance in the SA patients and an increase in neutrophils and eosinophils in DIF. SCF secreted by AFb from DIF was significantly higher than in SA. TNF + IL-1 significantly decreased the secretion of SCF by AFb. There was a positive correlation between SCF levels and the percentage eosinophils but not for metachromatic cells. Alpha-actin expression of AFb in DIF was significantly higher than in SA. Cytokine mRNA was extracted from AFb of two SA and two DIF patients. The profile showed that only in stimulated AFb isolated from the DIF patients can IL-5 transcripts be visualized. In conclusion, AFb can contribute to the onset of fibrosis by secreting SCF and IL-5 which, in turn, may recruit eosinophils.

Non-obese diabetic (NOD) mice spontaneously develop insulin-dependent (type 1) diabetes mellitus (IDDM) caused by T cells which destroy the insulin-producing islet β-cells. Since cytokines are involved in this auto-immune β-cell damage, we used an ELISPOT assay to enumerate the islet-associated T cells that secreted interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) or interleukin-4 (IL-4). We used mitogenic anti-CD3 antibody to activate all the T cells capable of responding, irrespective of their antigen specificity. We found that NOD females, more susceptible than males to IDDM, accumulated islet IFN-γ producers more rapidly with age than did the males. Acceleration of male IDDM by cyclophosphamide led to a marked increase in IFN-γ secreting islet T cells. In contrast, a decrease in IFN-γ-producing islet T cells was associated with arrest of IDDM by administration of peptide p277 of the 60kDa heat-shock protein (hsp60) to 12-week-old female NOD mice. The p277-treated mice later manifested a greater number of islets and fewer leukocytes per islet than did the mice treated with a bacterial hsp60 peptide. Thus, the development of diabetes could be correlated with the accumulation in the islets of T cells producing IFN-γ, and destructive insulitis could be downregulated by the administration of a single peptide.

Activated mast cells reside in close apposition to T cells in some inflammatory processes, In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release, Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells, Mast cells cocultured with activated T cells released histamine and beta-hexosaminidase and produced tumor necrosis factor alpha (TNF-alpha), an effect that peaked at 20 h, Kinetics of histamine release paralleled the formation of heterotypic aggregates, Separation of the two cell populations with a porous membrane prevented mediator release and TNF-alpha production, Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-alpha production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.

There has been substantial evidence that suggests that heparin may modulate various aspects of immune function and inflammation in addition to its well known anticoagulant activity. In this regard heparin was found to suppress cell-mediated immune responses or asthmatic reactions to allergen challenge. In the present study we analyse the effects of low molecular weight heparin (LMWH) on mast cell degranulation and cytokine production in vitro and on the elicitation of IgE-mediated mast cell-dependent late cutaneous allergic inflammation in vivo. We have established that LMWH preferentially inhibited tumour necrosis factor-alpha (TNF-alpha) and IL-4 production without having any significant effect on mast cell degranulation. These effects have been observed in mast cells derived from three different origins that were activated by either immunological or non-immunological stimuli. We have shown that there is inhibition of TNF-alpha production (and not neutralization of activity), as elimination of the drug after a short preincubation and addition of LMWH to rTNF-alpha had no effect on TNF-alpha-mediated cytotoxic activity. These results were also confirmed by ELISA. In vivo, s.c. injection of the LMWH inhibited the leucocyte infiltration associated with the late cutaneous response which followed passive cutaneous anaphylaxis (PCA) reaction, without affecting mast cell numbers or degranulation. These data suggest that LMWH may have an inhibitory role in mast cell-mediated allergic inflammation, and thus might be considered as a possible therapeutic modality.

We previously reported that immunity to the p277 peptide of the human 60-kDa heat shock protein (hsp60) was a causal factor in the diabetes of non-obese diabetic (NOD) mice, which are genetically prone to develop spontaneous autoimmune diabetes. The present study was done to test whether immunization with the p277 peptide could cause diabetes in standard strains of mice. We now report that a single administration of the p277 peptide conjugated to carrier molecules such as bovine serum albumin or ovalbumin can induce diabetes in C57BL/6 mice and in other strains not genetically prone to develop diabetes. The diabetes was marked by hyperglycemia, insulitis, insulin autoantibodies, glucose intolerance and low blood levels of insulin. The diabetes could be transferred to naive recipients by anti-p277 T cell lines. Similar to other experimentally induced autoimmune diseases, the autoimmune diabetes remitted spontaneously. After recovery, the mice were found to have acquired resistance to a second induction of diabetes. Susceptibility to induced diabetes in C57BL/6 mice was influenced by sex (males were much more susceptible than were females) and by class II genes in the major histocompatibility complex (B6.H-2(bm12) mice with a mutation in the MHC-II molecule were relatively resistant). Other strains of mice susceptible to induced diabetes were C57BL/KSJ, C3HeB/FeJ, and NON/Lt. BALB/c and C3H/HeJ strains were relatively resistant. Immunization to p277-carrier conjugates could also induce transient hyperglycemia in young NOD mice, but upon recovery from the induced diabetes, the NOD mice were found to have acquired resistance to later development of spontaneous diabetes. Thus, T cell immunity to the p277 peptide can suffice to induce diabetes in standard mice, and a short bout of induced diabetes can affect the chronic process that would otherwise lead to spontaneous diabetes in diabetes-prone NOD mice.

Airway inflammation is characterized by intraluminal influx of inflammatory cells, exudation of plasma, and increased procoagulant activity. We speculated that inflammatory cells might adhere to the airway surface epithelium in order to better localize and regulate airway inflammatory responses. Therefore, in this study, we asked whether neutrophils adhere to airway epithelial cells, whether serum or plasma factors increase adhesion, and, if so, what the characteristics of the involved adhesion molecules are. To answer these questions, we incubated human 51Cr-labeled neutrophils from peripheral blood with dog tracheal epithelial cells in culture in the presence or absence of normal human serum or plasma. After 30 min, nonadhering neutrophils were centrifuged away and neutrophil adhesion was assessed by radioassay. We found that unstimulated adhesion of neutrophils to cultured epithelial cells was quite low (< 6%). However, incubation with 10% serum or plasma increased adhesion of neutrophils to epithelial cells dramatically (up to a mean of 71%). The serum-induced increase in adhesion was concentration dependent; even 1% serum was effective (19% adhesion). Serum adhesion factor acted selectively on epithelial surfaces, was heat sensitive, had a molecular weight > 12,000, and depended on the presence of divalent cations. mAb 60.3 (anti-CD18) and mAb anti-Mol (anti-CD11b, anti-CR3) inhibited serum-induced adhesion by > 50% each. We conclude that normal serum and plasma contain a potent adhesion factor that induces adhesion of neutrophils to tracheal epithelium in culture.(ABSTRACT TRUNCATED AT 250 WORDS)