Papers by Gabriel Virella
Diabetes, 2000
We investigated the hypothesis that modified lipoproteins trigger an immune response leading to t... more We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential o...
Diabetes care, Jan 7, 2015
There is considerable interest in identifying biomarkers that predict high risk for the developme... more There is considerable interest in identifying biomarkers that predict high risk for the development of macrovascular complications in patients with diabetes. Therefore, the longitudinal association between subclinical atherosclerosis as measured by internal carotid artery intima-medial thickness (IMT) and acute-phase reactants, cytokines/adipokines, thrombosis, and adhesion molecules was examined. Biomarkers were measured at four time points over 20 years in 886 DCCT/EDIC participants with type 1 diabetes. Four composite scores were created by combining Z scores generated from within the dataset of individual biomarkers: acute-phase reactants (fibrinogen, C-reactive protein), thrombosis (fibrinogen, active and total plasminogen activator inhibitor [PAI]-1), cytokines/adipokines (tumor necrosis factor receptor-1 and -2, active and total PAI-I, IL-6), and endothelial dysfunction (soluble intracellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and soluble E-select...
Journal of lipid …, 2006
Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in human... more Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized ...
Atherosclerosis
OBJECTIVE: Circulating immune complexes (IC) containing modified forms of LDL (mLDL) are strongly... more OBJECTIVE: Circulating immune complexes (IC) containing modified forms of LDL (mLDL) are strongly pro-inflammatory and strong predictors of cardiovascular disease (CVD) progression in type 1 diabetes. The present study was undertaken to determine whether the levels of oxidized LDL (oxLDL), malondialdehyde-LDL (MDA-LDL) and advanced glycation end products-LDL (AGE-LDL) in IC predict incident CVD events in type 2 diabetes (VADT cohort). METHODS AND RESULTS: Levels of mLDL in IC were measured in 907 patients of the VADT cohort, a median of two years after entry into the study. Participants were followed for an average of 3.7 years for vascular outcomes. Hazard ratios (HRs) for CV endpoints in relation to mLDL-IC quartiles were calculated by Cox proportional hazard models. The primary composite CVD endpoint included documented myocardial infarction (MI); stroke; death from CVD; congestive heart failure; cardiac, cerebrovascular, or peripheral VD surgical intervention; inoperable CVD; an...
Nephrology Dialysis Transplantation
Modified low-density lipoprotein (LDL) immune complexes (IC) have proinflammatory properties and ... more Modified low-density lipoprotein (LDL) immune complexes (IC) have proinflammatory properties and play a role in albuminuria development. We measured oxidized LDL (oxLDL) and advanced glycation end-product (AGE)-LDL in IC isolated from sera of Type 1 diabetic subjects followed for 14-20 years and studied their association with abnormal albuminuria. Patients with albumin excretion rates (AER)<40 mg/24 h at baseline and follow-up (n=302) were deemed resistant to developing abnormal albuminuria. Patients with AER<40 mg/24 h at baseline whose AER levels progressed to >40 mg/24 h were considered prone to abnormal albuminuria (n=185), those who progress to AER>299 mg/24 h were considered as having macroalbuminuria (n=57). The odds of developing abnormal albuminuria were estimated by logistic regression based on natural log-transformed levels of oxLDL and AGE-LDL in IC and stratified by baseline AER decile. OxLDL and AGE-LDL were significantly higher in IC isolated from patients...
Atherosclerosis
Oxidized LDL (oxLDL) and oxLDL antibodies form immune complexes (IC) that reflect essential compo... more Oxidized LDL (oxLDL) and oxLDL antibodies form immune complexes (IC) that reflect essential components in the development of atherosclerosis: dyslipidemia, oxidative stress and induction of a pro-inflammatory humoral immune response. We measured oxLDL in IC (oxLDL-IC) isolated from patients with type 1 diabetes to assess the relationship between oxLDL-IC and coronary artery calcification (CAC). OxLDL was measured in IC isolated from baseline samples from a subgroup of 476 patients of the Diabetes Control and Complications Trial (DCCT). CAC was determined by computed tomography (CT) 11-20 years later. Multivariable log-binomial regression models were used to estimate the risk ratios associated with having a high CAC score with an increase of 1 standard deviation (SD) of the natural logarithm of oxLDL-IC. Multivariable regression models indicate that a 1 SD increase in the levels of oxLDL-IC was associated with a 37% increase in the risk of having high CAC score (RR=1.36; 95% CI: 1.12...
Diabetes Care
OBJECTIVE The current study aimed to determine in the Diabetes Control and Complications Trial (D... more OBJECTIVE The current study aimed to determine in the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications cohort whether or not abnormal levels of markers of inflammation and endothelial dysfunction measured in samples collected at DCCT baseline were able to predict the development of macroalbuminuria.RESEARCH DESIGN AND METHODS Levels of inflammation and endothelial cell dysfunction biomarkers were measured in 1,237 of 1,441 patients enrolled in the DCCT study who were both free of albuminuria and cardiovascular disease at baseline. To test the association of log-transformed biomarkers with albuminuria, generalized logistic regression models were used to quantify the association of increased levels of biomarkers and development of abnormal albuminuria. Normal, micro-, and macroalbuminuria were the outcomes of interest.RESULTSIn the logistic regression models adjusted by DCCT treatment assignment, baseline albumin excretion rate, ...
Journal of Immunoassay and Immunochemistry
Modified lipoproteins are able to induce inflammatory reactions through innate immunity pathways ... more Modified lipoproteins are able to induce inflammatory reactions through innate immunity pathways and are immunogenic, leading to an autoimmune response that results in the formation of proinflammatory immune complexes. The measurement of circulating oxidized lipoproteins and corresponding antibodies has, therefore, been proposed as an approach to assess the risk for complications in patients with diabetes and for the risk of cardiovascular disease in the general population. However, the majority of modified low density lipoprotein (LDL) in the peripheral circulation exists in the form of immune complexes, and this is a significant obstacle for the measurement of modified LDL and the corresponding antibodies. In this manuscript, we describe in detail the methodology developed by our group for isolation and fractionation of circulating immune complexes (IC), allowing the accurate assay of different LDL modifications. This approach has resulted in several studies showing that the level...
Diabetes care, Jan 7, 2015
There is considerable interest in identifying biomarkers that predict high risk for the developme... more There is considerable interest in identifying biomarkers that predict high risk for the development of macrovascular complications in patients with diabetes. Therefore, the longitudinal association between subclinical atherosclerosis as measured by internal carotid artery intima-medial thickness (IMT) and acute-phase reactants, cytokines/adipokines, thrombosis, and adhesion molecules was examined. Biomarkers were measured at four time points over 20 years in 886 DCCT/EDIC participants with type 1 diabetes. Four composite scores were created by combining Z scores generated from within the dataset of individual biomarkers: acute-phase reactants (fibrinogen, C-reactive protein), thrombosis (fibrinogen, active and total plasminogen activator inhibitor [PAI]-1), cytokines/adipokines (tumor necrosis factor receptor-1 and -2, active and total PAI-I, IL-6), and endothelial dysfunction (soluble intracellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and soluble E-select...
International journal of clinical & laboratory research, 1993
We have developed a solid-phase enzyme immunoassay for anti-oxidized low-density lipoprotein anti... more We have developed a solid-phase enzyme immunoassay for anti-oxidized low-density lipoprotein antibodies. Most sera showed some degree of non-specific binding to plates coated with oxidized low-density lipoprotein and the autoantibodies to oxidized low-density lipoprotein often appeared to have a relatively low affinity. To differentiate between specific and non-specific binding each sample was tested untreated and after absorption with oxidized low-density lipoprotein. The optical densities obtained with dilutions of the absorbed sample were considered to reflect non-specific binding and were subtracted from values obtained with identical dilutions of the unabsorbed sample, to yield corrected values from which the concentrations of anti-oxidized low-density lipoprotein antibody were calculated. Similar absorptions with native low-density lipoprotein and oxidized human serum albumin failed to induce a significant reduction in binding to immobilized oxidized low-density lipoprotein pr...
Metabolism, 2014
Sphingolipid metabolism is altered in diabetes and we analyzed the plasma concentrations of sphin... more Sphingolipid metabolism is altered in diabetes and we analyzed the plasma concentrations of sphingolipid species to investigate their association with the development of albuminuria in type 1 patients with diabetes. Samples were collected from 497 type 1 diabetic patients during their enrollment into the Diabetes Control and Complications Trial (DCCT). We determined plasma concentrations of multiple ceramide species and individual sphingoid bases and their phosphates using high performance liquid chromatography-tandem mass spectrometry and investigated their association with the development of albuminuria during 14-20 years of follow-up. Patients exhibited normal albumin excretion rates (AER &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;40 mg/24h) at the time of plasma sampling. Although the majority of patients (N = 291; 59%) exhibited normal levels of albuminuria throughout follow-up, 141 patients (28%) progressed to microalbuminuria (40 mg/24h ≤ AER&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;300 mg/24h), while 65 (13%) progressed to macroalbuminuria (AER ≥ 300 mg/24h). To test the association of log transformed plasma sphingolipid level with the development of albuminuria, generalized logistic regression models were used where normal, micro- and macroalbuminuria were the outcomes of interest. Models were adjusted for DCCT treatment group, baseline retinopathy, gender, baseline HbA1c %, age, AER, lipid levels, diabetes duration, and the use of ACE/ARB drugs. Increased plasma levels of very long, but not long chain ceramide species measured at DCCT baseline were associated with decreased odds to develop macroalbuminuria during the subsequent nineteen years (DCCT Baseline to EDIC year 8). These studies demonstrate, prospectively, that decreased plasma levels of select ceramide species are associated with the development of macroalbuminuria in type 1 diabetes.
Clinical Immunology and Immunopathology, 1991
We have studied the effects of dietary supplementation with fish oil on immunological parameters ... more We have studied the effects of dietary supplementation with fish oil on immunological parameters in a group of six normal volunteers, four of whom received a fish oil extract (total EPA dose of 2.4 g/day, which is on the lower range of clinically effective doses) for 6 weeks and two of which received a placebo (olive oil) for an identical period of time. Each volunteer was followed up for a period of 23 weeks after the dietary intervention was ended. All volunteers wei'e boosted witEtetanus toxoid (TI') at the onset of the trial. Several immune parameters were followed longitudinally, including NBT reduction and lysozyme release to test neutrophil function; lymphocyte subpopulations; mitogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and anti-CD3; IL-2 release after PHA and pokeweed mitogen (PWM) stimulation; immunoglobulin and anti-TT antibody (ATr) synthesis by stimulated lymphocytes; and serum levels of immunoglobulins and of ATT. No consistent changes were observed in neutrophil function tests, mitogenic responses to PHA and Con A, and lymphocyte subsets. The mitogenic response to anti-CD3 and the release of IL-2 after stimulation with PHA and PWM appeared reduced as a consequence of fish oil ingestion, and levels of serum immunoglobulins decreased in three of the volunteers receiving fish oil supplementation. The systemic humoral response after the "IT booster appeared not to be influenced by the ingestion offish oil. However, in those subjects who were given fish oil supplementation, the specific in vitro response of their peripheral blood lymphocytes to TT appeared to be compromised at Week 3. This could reflect the need for progressive accumulation of EPA in lymphocyte membranes for the suppressive effect to be detectable, but it could also reflect a differential sensitivity to the effects of fish oil of circulating B lymphocytes vs. bone marrow B lymphocytes. All the parameters apparently affected by fish oil ingestion were also affected by the incubation of normal lymph_ocytes with EPA in vitro.
Clinical Immunology and Immunopathology, 1997
whose atherogenic potential appears to be related to Antibodies to oxidized LDL (ox-LDL) and LDL-... more whose atherogenic potential appears to be related to Antibodies to oxidized LDL (ox-LDL) and LDL-conthe presence of LDL and antibodies of the IgG and IgA taining immune complexes (LDL-IC) have been reisotypes. ᭧ 1997 Academic Press ported to be associated with the presence or progression of arteriosclerosis. We screened for anti-modified INTRODUCTION LDL antibodies and isolated soluble IC by precipitation with 3.5% (w/v) polyethylene glycol (PEG) 6000 in two groups. The patient group was constituted by 16
Diabetes / Metabolism Reviews, 1996
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Immunochemistry, 1978
purified IgA proteins were typed for subciass and allorype serologically and immunochemically. Sp... more purified IgA proteins were typed for subciass and allorype serologically and immunochemically. Spontaneous release of light chain dimers was studied in correlation with susceptibility to streptococcal protease and, in a smaller group, the ability to interact with staphylococcal protein A, As a rule serological and immunochemical typing agreed, but the results differed for two proteins. In one case the discrepancy could be attributed to contaminatjon of an IgAl protein with lgA2 A2m (1) molecules or, more likely, to the existence of an fg4.2 A2m (1) protein with d~sulfide bonds joining the heavy and light chains. The second case was apparently a mistyping, since the protein behaved as IgAl by all immunochemical criteria and the reaction with anti-IgA2 A2m (i) was weak. Upon incomplete reduction of interchain bonds, most of the IgAl proteins and all the IgA2 AZm (1) proteins released predominantly H2 subunits; but in a small number of IgAl proteins, release of ML subunits was equal to or greater than the release of H2, suggesting that additional serotypes of IgA remain to be defined.
Journal of Diabetes and its Complications, 2011
Immune complexes containing modified LDL (LDL-IC) and NMR-determined total LDL particle concentra... more Immune complexes containing modified LDL (LDL-IC) and NMR-determined total LDL particle concentrations are significantly associated with intima-medial thickness (IMT). We analyzed the associations between concentrations of NMR-determined lipoprotein subclasses and LDL-IC in the DCCT/EDIC cohort. LDL-IC concentrations in women and men of the DCCT/EDIC cohort did not differ significantly and were positively associated with Total LDL particle concentrations in men and women (r=0.34, r=0.32, respectively; p<0.01) and with Small LDL concentration (r=0.22, r=0.13, respectively; p<0.01). In women, Large LDL concentrations were also associated with LDL-IC (r=0.20, p<0.01) while in men, the association was more modest (r=0.11, p<0.05). Thus, both Small and Large LDL are associated with LDL-IC formation. Based on the results from statistical mediation analyses, we concluded that plasma concentrations of LDL-IC may provide a physiological link between the statistically significant association of Total LDL particle concentration with carotid artery intima-media thickness in subjects with Type 1 diabetes. Furthermore, after adjusting for conventional risk factors, there was a decrease in LDL-IC concentration even in the presence of high Total LDL particle concentrations in those women with high concentrations of Large HDL but the association was not evident in men. This suggests that the associations between Large HDL and Total LDL particle concentrations, and their associations with LDL-IC levels, differ by gender and suggest that LDL-IC partially mediates the contribution of Total LDL particle concentration to increased carotid IMT in diabetic men.
Immunology, 2012
Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contrib... more Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcc receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was upregulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1b-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.
Diabetes Care, 2008
OBJECTIVE -Because endothelial cell dysfunction and inflammation are key contributors to the deve... more OBJECTIVE -Because endothelial cell dysfunction and inflammation are key contributors to the development of complications in type 1 diabetes, we studied risk factors related to endothelial dysfunction and inflammation (C-reactive protein and fibrinogen, soluble vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin, and fibrinolytic markers) in a subgroup of patients from the Diabetes Control and Complications Trial (DCCT)/ Epidemiology of Diabetes Intervention and Complications (EDIC) study cohort.
Clinical Immunology, 2008
Modified lipoproteins are immunogenic and play a key pathogenic role in vascular disease. Antibod... more Modified lipoproteins are immunogenic and play a key pathogenic role in vascular disease. Antibodies to oxidized LDL (oxLDL) are mostly of the proinflammatory IgG 1 and IgG 3 isotypes. We measured IgG and IgM oxLDL antibodies in immune complexes (IC) isolated from 36 patients with type 1 diabetes using a nested case-control design. IgG antibodies predominated over IgM antibodies by an 8:1 ratio. IgG antibody concentrations were higher in the nephropathy cases compared to controls (p=0.09), but no significant difference was observed because of two patients included in the study who had end-stage renal disease (creatinine > 5mg/dl and glomerular filtration rate (GFR) less than 17 ml/min). After eliminating these patients from the analysis, significant positive associations of IgG antibody concentration with serum creatinine and albumin excretion rate were observed after eliminating two patients with significant renal impairment (serum creatinine > 5 mg/dl). Similarly, a negative correlation with estimated glomerular filtration rate was observed in this subsample of 34 patients. Differences in IgM antibody concentrations by nephropathy classification were not supported by the data. In conclusion, the predominance of pro-inflammatory IgG oxLDL antibodies is associated with existence of diabetic nephropathy, and a protective role of IgM antibodies could not be demonstrated.
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Papers by Gabriel Virella