Patients with autoimmune chronic inflammatory diseases present increased biomarkers of thromboinflammation and endothelial dysfunction in the absence of flares and cardiovascular comorbidities.

Cardiovascular risk is increased in patients with autoimmune rheumatic diseases. Endothelial, erythrocyte and platelet microvesicles (MVs) are elevated in patients with cardiovascular diseases and represent novel markers of endothelial dysfunction and thromboinflammation. We tested whether their levels are increased in patients with autoimmune rheumatic diseases (ARDs) in the absence of disease flare and cardiovascular comorbidities. Well-controlled patients with rheumatoid arthritis or systemic lupus erythematosus were studied, provided they were free from cardiovascular comorbidities and established cardiovascular disease. We additionally studied (a) a control group consisting of healthy volunteers and (b) a reference group including patients with stable coronary artery disease (CAD). MVs were measured using a standardized flow cytometry protocol. In a population of 74 participants, patients with ARDs (n = 17) presented increased levels of both endothelial (283.3 ± 195.0/µL vs 168.5 ± 54.8/µL, p = 0.029) and platelet MVs (374.0 ± 275.3/µL vs 225.7 ± 101.1/µL, p = 0.046) compared to controls (n = 34), whereas erythrocyte MVs did not significantly differ. In addition, patients with ARDs showed similar levels of endothelial MVs compared to CAD patients (n = 23) (283.3 ± 195.0/µL vs 297.0 ± 211.8/µL, p = 0.846). Platelet MVs were significantly associated with disease duration, and erythrocyte MVs with patients' perceived disease activity. In conclusion, increased levels of endothelial and platelet MVs may be evident in patients with ARDs, even in the absence of disease flares and before the establishment of cardiovascular complications. Levels of endothelial MVs resemble those of patients with profound atherothrombotic profile. The prognostic potential of MVs in terms of cardiovascular disease prevention warrants further investigation in patients with ARDs.

Assessment of Endothelial Injury and Pro-Coagulant Activity Using Circulating Microvesicles in Survivors of Allogeneic Hematopoietic Cell Transplantation.

(1) Background: survivors of allogeneic hematopoietic cell transplantation (alloHCT) suffer from morbidity and mortality due to cardiovascular events. We hypothesized that vascular injury and pro-coagulant activity are evident in alloHCT survivors without existing alloHCT complications or relapse. (2) Methods: we enrolled consecutive adult alloHCT survivors without established cardiovascular disease and control individuals matched for traditional cardiovascular risk factors (January-December 2019). Circulating microvesicles (MVs) of different cellular origins (platelet, erythrocyte, and endothelial) were measured by a standardized flow cytometry protocol as novel markers of vascular injury and pro-coagulant activity. (3) Results: we recruited 45 survivors after a median of 2.3 (range 1.1-13.2) years from alloHCT, and 45 controls. The majority of patients suffered from acute (44%) and/or chronic (66%) graft-versus-host disease (GVHD). Although the two groups were matched for traditional cardiovascular risk factors, alloHCT survivors showed significantly increased platelet and erythrocyte MVs compared to controls. Within alloHCT survivors, erythrocyte MVs were significantly increased in patients with a previous history of thrombotic microangiopathy. Interestingly, endothelial MVs were significantly increased only in alloHCT recipients of a myeloablative conditioning. Furthermore, MVs of different origins showed a positive association with each other. (4) Conclusions: endothelial dysfunction and increased thrombotic risk are evident in alloHCT recipients long after alloHCT, independently of traditional cardiovascular risk factors. An apparent synergism of these pathophysiological processes may be strongly involved in the subsequent establishment of cardiovascular disease.

Evaluation of regulatory T cells (Tregs) alterations in patients with multiple myeloma treated with bortezomib or lenalidomide plus dexamethasone: correlations with treatment outcome.

The exact role of regulatory T cells (Tregs) in multiple myeloma (MM) has not been yet determined. Data regarding alterations of Tregs during therapy with novel agents (NA), i.e., bortezomib and lenalidomide are conflicted and limited. We evaluated prospectively alterations of Tregs and searched for correlations with disease characteristics, response, and outcome in 29 patients with active MM treated with either bortezomib-dexamethasone (BD; 11 patients) or lenalidomide-dexamethasone (LenDex, 18 patients). Additionally, we recorded changes of lymphocytes subsets and cytokines related to Tregs function and MM biology, i.e., interleukin (IL) 6, 2, 17, and TGF-ß. Compared with controls, patients had significantly higher median levels of Tregs%, IL-6, and IL-17 (p < 0.001). Median CD4 T and B cells frequencies were significantly lower, whereas CD8 T and natural killers were increased compared to controls. In BD group, no significant alterations of Tregs% were observed. Patients treated with LenDex, displayed a significant reduction of Tregs% (p < 0.001) especially those who achieved at least very good partial response (≥vgPR) (p = 0.04). Lymphocyte subsets or cytokines did not significantly change during therapy. In summary, Tregs% are higher in patients with active MM compared with controls, and they significantly decrease after treatment with LenDex but not with BD; After therapy with LenDex, Tregs reduction between baseline and major response correlated with achievement of ≥vgPR suggesting a possible predictive role, that may contribute to therapeutic strategy.

Assessment of microvesicles from different cell origins in patients with psoriasis: evidence of thrombogenic, proinflammatory microenvironment in the absence of established cardiovascular disease.

Psoriasis is associated with increased cardiovascular risk. Endothelial, platelet, and erythrocyte microvesicles (MVs) are novel biomarkers of endothelial dysfunction and thromboinflammation. We explored whether MVs of different cell types are elevated in patients with psoriasis, and investigated potential associations with disease severity and macrovascular function. Endothelial, platelet and erythrocyte MVs were measured using a standardized flow cytometry protocol in psoriasis patients and controls free from established cardiovascular disease. Carotid intima-media thickness (IMT) and pulse wave velocity (PWV) were measured as markers of subclinical atherosclerosis and arterial stiffness. Psoriasis severity was assessed with PASI (Psoriasis Area Severity Index). Both platelet (p < 0.001) and erythrocyte MVs (p = 0.046), yet not endothelial MVs, were significantly increased in patients with psoriasis (n = 41) compared with controls (n = 41). Patients with higher PASI (≥10) presented significantly higher levels of ErMVs compared to those with lower PASI (<10) (p = 0.047). Carotid IMT and PWV were comparable between psoriasis patients and controls and did not significantly correlate with MVs. In the multivariate analysis, psoriasis was identified as an independent predictor of both platelet (p < 0.001) and erythrocyte MVs (p = 0.043), while hypertension was independently associated with endothelial MVs (p < 0.001). Increased formation of platelet and erythrocyte MVs may be evident in psoriasis patients and is indicative of prothrombotic, proinflammatory microenvironment, even in the absence of subclinical macrovascular dysfunction and before the clinical onset of overt cardiovascular complications. Potential mechanistic links and prognostic implications of increased MVs in psoriasis warrant further investigation.

Circulating microvesicles across a population with various degree of cardiovascular burden are associated with systolic blood pressure.

Circulating microvesicles (MVs) have been studied in heterogeneous, divergent, and rather small patient populations with cardiovascular risk . Therefore, we measured endothelial (EMVs), platelet (PMVs) and erythrocyte (RMVs) MVs in patients with divergent cardiovascular risk. We then compared them to coronary artery disease (CAD) and healthy subjects and identified independent MVs' predictors. We enrolled consecutive patients from our Cardiology, Hypertension, Diabetic, Rheumatic, and Nephrology Outpatient Units with MVs measurements. Central blood pressure (BP) was measured by either applanation tonometry or Mobil-O-graph device, while MVs by a standardized flow cytometry protocol. We studied 369 participants with increased cardiovascular risk: 63 with high cardiovascular risk (47 diabetes mellitus type II/DM and 16 end-stage renal disease/ESRD), 92 with chronic inflammatory disorders and 73 with untreated essential hypertension/UEH. We further included 53 subjects with CAD and 87 otherwise healthy individuals. All MVs were lower in patients with increased cardiovascular risk compared to CAD, showing predictive value with high sensitivity and specificity. Furthermore, PMVs and EMVs were increased in patients with cardiovascular risk compared to healthy individuals. DM and ESRD patients had increased EMVs versus UEH and chronic inflammatory disorders. In the whole study population, RMVs were associated only with history of essential hypertension. In multivariate analysis, systolic BP predicted PMVs. Aage, systolic BP, and DM predicted EMVs. In a large population of patients with divergent cardiovascular risk, MVs are independently associated with systolic blood pressure.

Increased erythrocyte- and platelet-derived microvesicles in newly diagnosed type 2 diabetes mellitus.

AIM: To investigate the thrombotic microenvironment in early stages of type 2 diabetes mellitus measuring platelet-derived, endothelial-derived and erythrocyte-derived microvesicles. METHODS: We recruited 50 newly diagnosed type 2 diabetes mellitus patients who did not receive glucose-lowering treatment except for metformin and 25 matched non-type 2 diabetes mellitus volunteers. Microvesicles were measured with flow cytometry, glycated haemoglobin with high-performance liquid chromatography and advanced glycation end products with enzyme-linked immunosorbent assay. RESULTS: Type 2 diabetes mellitus patients showed significantly higher levels of platelet-derived microvesicles [195/µL (115-409) vs 110/µL (73-150), p = 0.001] and erythrocyte-derived microvesicles [26/µL (9-100) vs 9/µL (4-25), p = 0.007] compared to non-type 2 diabetes mellitus individuals. Platelet-derived microvesicles were positively associated with fasting blood glucose (p = 0.026) and glycated haemoglobin (p = 0.002). Erythrocyte-derived microvesicles were also positively associated with fasting blood glucose (p = 0.018) but not with glycated haemoglobin (p = 0.193). No significant association was observed between platelet-derived microvesicles (p = 0.126) or erythrocyte-derived microvesicles (p = 0.857) and advanced glycation end products. Erythrocyte-derived microvesicles predicted the presence of type 2 diabetes mellitus, independently of platelet-derived microvesicles. CONCLUSION: In newly diagnosed type 2 diabetes mellitus, ongoing atherothrombosis is evident during the early stages as evidenced by increased microvesicles levels. Furthermore, the association with glycemic profile suggests that microvesicles represent not only a novel mechanism by which hyperglycemia amplifies thrombotic tendency in type 2 diabetes mellitus but also early markers of thrombosis highlighting the need for earlier management of hyperglycemia.

Endothelial Microvesicles Circulating in Peripheral and Coronary Circulation Are Associated With Central Blood Pressure in Coronary Artery Disease.

BACKGROUND: Endothelial microvesicles (EMVs) have emerged as markers of endothelial injury. However, little is known about their levels in the coronary circulation of acute coronary syndrome (ACS) and stable coronary artery disease (CAD). We hypothesized that ACS patients exhibit a more pronounced increase of EMVs both in the peripheral and coronary circulation when compared with CAD. We also investigated possible associations of EMVs with markers preclinical target organ damage. METHODS: We enrolled consecutive eligible patients undergoing coronary angiography. Blood samples were collected from the stem of the left coronary artery and the femoral artery. ΕMVs were measured by a standardized flow cytometry protocol. Central systolic blood pressure (cSBP) was measured invasively and patients' history was recorded. RESULTS: CAD patients exhibited increased levels of EMVs compared with controls. When patients with ACS and stable CAD were compared, the former had significantly increased EMVs in both coronary and peripheral circulation. Importantly, both ACS and CAD patients exhibited increased levels of EMVs in the coronary circulation compared with periphery. In addition, EMVs were associated with cSBP. CONCLUSIONS: EMVs emerge as novel markers of ongoing underlying vascular damage, further augmenting the vicious cycle of inflammation and thrombosis mainly in ACS but also in stable CAD.

Prevalence of coeliac disease in the adult population of central Greece.

OBJECTIVES: Recent studies from several countries have shown that coeliac disease (CD) is increasingly being diagnosed in adults, as the availability of new, accurate serologic tests has made screening in the general population possible. No data exist regarding the prevalence of CD in Greece. The aim of this study was the implementation of a serologic screening procedure for CD in the adult general population of Thessaly, an area of central Greece, using a novel diagnostic algorithm. METHODS: The study included 2230 participants (1226 women, 1004 men, median age 46 years, range 18-80 years), selected by systematic random sampling, from the adult general population of Thessaly. All the serum samples were tested for total immunoglobulin A (IgA)-serum levels, to exclude IgA deficiency. Samples with total IgA within the normal range were tested for IgA antibodies against native human-tissue transglutaminase (anti-tTG); samples that were anti-tTG positive were tested for IgA antiendomysial antibodies (EmA). Samples from participants with selective IgA deficiency were examined for IgG antigliadin antibodies. Participants who were EmA-positive or antigliadin antibody-positive were referred for intestinal biopsy and human leucocyte antigen (HLA) typing. RESULTS: No participant with selective IgA deficiency was detected. Four individuals tested positive for EmA, all of whom were biopsy-proven coeliacs. Therefore, the CD prevalence in this general population sample is 1 : 558 or 1.8 per 1000 (SE 0.13). The four new patients with abnormal histology (two men, two women) were aged between 18 and 35 years. Two of them were considered to be asymptomatic and two presented with a subclinical course. All four had the heterodimer HLA-DQ2. CONCLUSIONS: This first serological screening study for CD in Greece has demonstrated that CD prevalence in Thessaly is among the lowest reported in Europe.

Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases.

The prevalence of celiac disease (CD) and the prevalence and clinical significance of anti-tissue transglutaminase (tTG) antibodies (tTGAbs) in a large series of patients with chronic liver diseases were assessed. We studied 738 patients (462 with chronic viral hepatitis, 117 with autoimmune liver diseases, 113 with alcoholic or nonalcoholic fatty liver disease, and 46 with other liver disorders) and 1,350 healthy controls (HC). Immunoglobulin A (IgA) tTGAbs were measured by enzyme-linked immunosorbent assay and a microsphere-based flow cytometric assay. Positive sera were investigated for IgA antiendomysial antibodies (EmA). IgA tTGAb-positive subjects were invited to undergo a small-intestinal biopsy and HLA-DQ allele typing. Four of 1,350 HC (0.3%) tested tTGAb(+) EmA(+) and underwent a biopsy (CD confirmation in all). Four of 738 liver disease patients tested tTGAbs(+) EmA(+) (0.54%; not statistically significant). Two were HCV infected (1.24%; not statistically significant), and two had transaminasemia of unknown origin. Forty-three patients tested tTGAbs(+) EmA(-) (5.8%; P<0.001 compared to HC). Inhibition experiments verified the existence of specific IgA anti-tTG reactivity. Twenty-six of 43 patients underwent a biopsy (all negative for CD). Binary logistic regression analysis revealed age (P=0.008), cirrhosis (P=0.004), alkaline phosphatase (P=0.026), and antinuclear antibodies (P=0.012) as independent risk factors for tTGAb reactivity among the patients. It was concluded that CD prevalence is the same in HC and patients with chronic liver diseases. The prevalence of tTGAbs is higher in hepatic patients compared to HC, but their specificity for CD diagnosis in this group of patients is low. tTGAbs in patients appear to be associated with the presence of autoimmunity, cirrhosis, and cholestasis, irrespective of the origin of the liver disease.

Evaluation of a microsphere-based flow cytometric assay for diagnosis of celiac disease.

The multiplexed particle-based flow cytometric technology proposes a new approach for the diagnosis of autoimmune diseases combining the advantages of conventional methods with the ability to quantitatively determine multiple autoantibodies in the same sample, simultaneously and rapidly. Recently, a commercial kit (FIDIS Celiac, Biomedical Diagnostics, Mane la Vallé, France) was introduced for the simultaneous detection of IgA anti-tissue transglutaminase (anti-tTG), IgG, and IgA anti-gliadin antibodies (AGA). This study was undertaken to evaluate and compare the FIDIS Celiac kit with standardized commercial ELISAs (QUANTA Lite, INOVA Diagnostics Inc., San Diego, CA). A disease group consisted of 21 samples from untreated patients with biopsy confirmed celiac disease (CD), and two control groups of historical sera (207 from regular blood donors and 181 from chronically infected hepatitis patients) were studied. All control sera were negative for IgA anti-endomysial antibodies (EmA) and had an IgA concentration above the lower normal limit. Concerning the reproducibility, intra- and inter-assay coefficients of variation (CVs) ranging between 2% and 12%, and between 3% and 21%, respectively, were observed. Regarding the diagnostic quality, each assay was compared to the disease diagnosis using the McNemar test and the kappa (K) parameter, while ROC analysis was applied. Generally, the performance of FIDIS assay was proved almost equally adequate to that of ELISA in the detection of IgA anti-tTG antibodies, IgA and IgG AGA. However, the performance of FIDIS assay was found surmounting that of ELISA among hepatitis patients, possibly due to the avoidance of debris and unbound cross contaminants and, hence, the "noise" of such materials in samples under analysis. Taking our results together with the simplicity and the high throughput of FIDIS assay, its overall performance in the diagnosis of CD seems better than that of ELISA.