Boris Gilburd

Risk factors for subclinical atherosclerosis in a prospective cohort of patients with systemic lupus erythematosus.

Objective: To evaluate traditional and non-traditional risk factors for subclinical atherosclerosis in systemic lupus erythematosus (SLE). Methods: A prospective cohort of 78 patients with SLE without overt atherosclerotic disease was studied. SLE clinical and laboratory parameters, disease activity and damage, treatment and traditional risk factors for atherosclerosis were evaluated. At baseline (T1) and after five years’ follow up (T2), the serum levels of anti-oxidised palmitoyl arachidonoyl phosphocholine (oxPAPC), anti-heat shock protein 65, and anti-β2-glycoprotein I antibodies and C reactive protein were tested. At T2, intima-media thickness (IMT) was measured using duplex carotid sonography. Thickened intima, plaque, mean IMT (m-IMT), and maximum IMT (M-IMT) were assessed. Results: A thickened intima was seen in 22/78 (28%) patients and plaque in 13/78 (17%). M-IMT and m-IMT were (mean (SD)) 0.77 (0.34) mm and 0.55 (0.15) mm, respectively. Patients with carotid abnormalities were significantly older, had higher blood pressure and total serum cholesterol levels, and had taken a higher prednisone cumulative dosage than those without any lesions. The carotid abnormalities were associated with renal disease and ECLAM >2 at T1, and with azathioprine treatment. In multivariate analysis, age and cumulative prednisone dose were associated with carotid abnormalities; age, hypertension, and anti-oxPAPC at T2 were correlated with higher M-IMT and m-IMT. Conclusions: In patients with SLE some non-traditional risk factors for atherosclerosis were identified, the most important of which was the cumulative prednisone dose. The role of some traditional risk factors, such as age and hypertension, was also confirmed. The predictive value of the new immunological and inflammatory markers of atherosclerosis seems to be masked by some disease related features.

Hyperimmunization of apo-E-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early atherogenesis

The role of the immune system in modulating atherosclerosis has recently been the subject of intensive research. Several previous authors have put forward a paradigm of the autoimmune process occurring in the vicinity of the plaque. Two recent studies have shown that immunization of rabbits with homologous modified low-density lipoprotein (LDL) led to suppression of atherosclerosis. In the current study we evaluated the effects of homologous malondialdehyde (MDA)-LDL immunizations on atherogenesis in apo-E-deficient mice. Two groups of female chow-diet-fed, apo-E-deficient mice (n = 10) were either immunized with homologous MDA-LDL or with phosphate buffer saline (PBS) at 2-week intervals. The mice were sacrificed 12 weeks following the primary immunization. The MDA-LDL-immunized mice were shown to develop high titers of anti-MDA-LDL antibodies. Atherosclerosis, determined by the lesion size at the aortic sinus, was significantly suppressed in the MDA-LDL-immunized mice as compared with their littermates immunized with PBS (mean area +/- S.D.; 74000 +/- 17300 microm2 versus 158000 +/- 12800 microm2; P < 0.01). No differences were found between the groups with respect to the cellular composition of the atherosclerotic plaques. The results of this study show that immunization with MDA-LDL has a protective effect in apo-E-deficient mice, and further suggests that this mouse model is suitable for studies of immunomodulation.

Immunolocalization of β2-Glycoprotein I (Apolipoprotein H) to Human Atherosclerotic Plaques Potential Implications for Lesion Progression

Background—β2-Glycoprotein I (β2GPI) is a major antigenic target of antiphospholipid antibodies, which possesses natural anticoagulant properties. The aim of the present study was to determine its ...

Induction of Early Atherosclerosis in LDL-Receptor–Deficient Mice Immunized With β2-Glycoprotein I

BACKGROUND Immunization with beta2-glycoprotein I (beta2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of beta2GPI immunization on the progression of atherosclerosis. METHODS AND RESULTS In the first experiment, 3 groups of LDL receptor-deficient (LDL-RD) mice (n=15 per group) were immunized with either beta2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies as well as a specific lymph node proliferation to beta2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the beta2GPI-immunized mice (mean aortic lesion, 26 000+/-5700 microm2) in comparison with their ovalbumin-immunized (mean, 3000+/-1099 microm2; P<0.01) and nonimmunized (mean, 2250+/-700 microm2; P<0.01) littermates. The average lesion size in the beta2GPI-immunized mice fed an atherogenic diet (mean, 98 000+/-8305 microm2) was larger than the ovalbumin-immunized mice (mean, 81 250+/-12 933 microm2; P=NS) or the nonimmunized controls (mean, 75 625+/-7281 microm2; P=NS). The atherosclerotic plaques in the beta2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice. CONCLUSIONS The results of the present study provide the first direct evidence for the proatherogenic effect of ss2GPI immunization and establish a new model for immune-mediated atherosclerosis.

Enhanced Fatty Streak Formation in C57BL/6J Mice by Immunization With Heat Shock Protein-65

Recent data suggest that the immune system is involved in atherogenesis. Thus, interest has been raised as to the possible antigens that could serve as the initiators of the immune reaction. In the current work, we studied the effects of immunization with recombinant heat shock protein-65 (HSP-65) and HSP-65-rich Mycobacterium tuberculosis (MT) on early atherogenesis in C57BL/6J mice fed either a normal chow diet or a high-cholesterol diet (HCD). A rapid, cellular immune response to HSP-65 was evident in mice immunized with HSP-65 or with MT but not in the animals immunized with phosphate-buffered saline (PBS) alone. Early atherosclerosis was significantly enhanced in HCD-fed mice immunized with HSP-65 (n=10; mean aortic lesion size, 45 417+/-9258 microm2) or MT (n=15; 66 350+/-6850 microm2) compared with PBS-injected (n=10; 10 028+/-3599 microm2) or nonimmunized (n=10; 9500+/-2120 microm2) mice. No fatty streak lesions were observed in mice fed a chow diet regardless of the immunization protocol applied. Immunohistochemical analysis of atherosclerotic lesions from the HSP-65- and MT-immunized mice revealed infiltration of CD4 lymphocytes compared with the relatively lymphocyte-poor lesions in the PBS-treated or nonimmunized mice. Direct immunofluorescence analysis of lesions from HSP-65- and MT-immunized mice fed an HCD exhibited extensive deposits of immunoglobulins compared with the fatty streaks in the other study groups, consistent with the larger and more advanced lesions found in the former 2 groups. This model, which supports the involvement of HSP-65 in atherogenesis, furnishes a valuable tool to study the role of the immune system in atherogenesis.

Oral tolerance with heat shock protein 65 attenuates Mycobacterium tuberculosis-induced and high-fat-diet-driven atherosclerotic lesions.

OBJECTIVE The goal of this study was to explore the efficacy of oral tolerance with heat shock protein (HSP) 65 in two apparently non-overlapping models of murine atherosclerosis. BACKGROUND Atherosclerosis is considered to be a chronic inflammatory process. Autoimmune mechanisms have been shown to influence atherogenesis in experimental animal models. Heat shock protein 65 is a candidate antigen thought to drive a proatherogenic immune-mediated response. Mucosal tolerance is a therapeutic means of accomplishing immune unresponsiveness toward a given antigen by feeding it before active induction of the disorder. METHODS Low-density lipoprotein receptor deficient mice were fed with different doses of HSP65 every other day for 10 days. Feeding with either bovine serum albumin (BSA) or phosphate buffered saline (PBS) served as control. One day after the last feeding, mice were challenged either by immunization with heat killed Mycobacterium tuberculosis or by a high fat diet. RESULTS Lymphocyte reactivity from mice fed with HSP65 and immunized either against HSP65 or M. tuberculosis was significantly reduced in comparison with BSA-fed mice. Moreover, co-incubation of splenocytes-from mice with tolerance induced with HSP65 but not BSA-with HSP65-reactive lymphocytes resulted in the suppression of HSP65 reactivity by the latter cells. Interleukin-4 production by HSP65-fed and immunized mice was increased upon priming with respective protein. Early atherosclerosis was attenuated in HSP65-fed mice, compared with either BSA- or PBS-fed mice, regardless of the method employed to induce fatty streaks (M. tuberculosis immunization or high-fat diet). CONCLUSIONS Oral tolerance induced with HSP65 could prove to be a novel means of suppressing atherogenesis.

Adoptive Transfer of β2-Glycoprotein I–Reactive Lymphocytes Enhances Early Atherosclerosis in LDL Receptor–Deficient Mice

Background—It has been proposed that autoimmune factors can influence the progression of atherosclerosis. We have previously shown that immunization of LDL receptor–deficient (LDL-RD mice) with β2-glycoprotein I (β2GPI; a principal target of “autoimmune” antiphospholipid antibodies) enhances early atherosclerosis. In the present study, we tested the hypothesis that adoptive transfer of β2GPI-reactive T cells can accelerate fatty streak formation in LDL-RD mice. Methods and Results—LDL-RD mice were immunized with human β2GPI. An additional group of mice were immunized with β2GPI and boosted with the same antigen 3 weeks later. Control mice with immunized with human serum albumin. Lymphocytes obtained from the draining lymph node cells or from splenocytes of β2GPI- or human serum albumin–immunized mice were stimulated in vitro with β2GPI or with the mitogen concavalin A, respectively. The cultured lymphocytes were transferred intraperitoneally to syngenic LDL-RD mice, and the mice were fed a high-fat “Weste...

Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice

OBJECTIVES This study was designed to determine the role of cellular and humoral immune responses to heat shock protein 65 (HSP65) in murine atherosclerosis. BACKGROUND Inflammatory processes appear to influence the progression of atherosclerosis. Immunization with HSP65 was previously shown to induce arteriosclerosis in rabbits and to enhance fatty-streak formation in mice. However, it has not been demonstrated directly whether HSP65-reactive antibodies and lymphocytes are separately capable of influencing lesion formation. METHODS Low density lipoprotein-receptor deficient (LDL-RD) mice were immunized with HSP65 or control bovine serum albumin (BSA). Lymph-node cells, splenocytes and immunoglobulin G (IgG) were obtained from the immunized mice and transferred separately to six groups of syngenic LDL-RD mice. RESULTS Adoptive transfer of HSP65-reactive lymph node cells increased fatty-streak formation in comparison with mice treated with BSA-primed cells. Similarly, transfer of splenocytes reactive with HSP65 led to enhanced fatty-streak generation compared with mice injected with BSA-sensitized splenocytes. Repeated intraperitoneal administration of IgG from serum of HSP65-immunized mice (every 10 days) enhanced fatty-streak formation in mice in comparison with their anti-BSA-IgG injected littermates. CONCLUSIONS Antibodies and lymphocytes reactive to HSP65 promote fatty-streak formation in mice, providing direct evidence for the proatherogenic properties of cellular and humoral immunity to HSP65.

Evaluation of the BioPlex 2200 ANA screen analysis of 510 healthy subjects : Incidence of natural/predictive autoantibodies

Abstract: The BioPlex™ 2200 ANA Screen is a fully automated system that determines levels for 13 different autoimmune antibodies of established clinical significance. The objective of this study was to determine the specificity of the BioPlex™ 2200 ANA Screen assay and to analyze the antibody profile samples collected from healthy subjects against comparative ELISA and IIF screening methods. A total of 510 specimens were randomly selected from a cohort of apparently healthy blood bank donors. Samples were distributed to five age brackets. All samples were tested using Bio‐Rads ANA Screen kit. Specificity was compared to IIF and ELISA results. Most of the samples were found negative in all ANA screening systems (84.5% by IIF, 92.5% by BioPlex™ 2200 ANA Screen kit, and 94.5% by ELISA). The frequency of positive results was highest (15.5%) using IIF, in comparison to almost similar results (5.5% vs. 7.5%) achieved by ANA ELISA and BioPlex™ 2200 ANA Screen kits. The positive rate of autoantibodies was significantly reduced when analyzed by different combinations of ANA screen assays (from 2.35% using IIF + BioPlex ANA Screen tests to 0.98% by using all three tests). Using the BioPlex™ 2200 ANA Screen system, we were able to identify samples with high levels of individual antibodies: anti‐dsDNA at 20‐63/IU/mL, antichromatin at 4–8 AI, anti‐SmRNP at 2–6 AI, and anti‐RNPA at 2‐4.5 AI. Importantly, from 7 IIF and ELISA positive sera, 5 of these were also BioPlex 2200 positive, suggesting that the BioPlex is seeing the samples that are of the greatest interest, using the established techniques. The specificity of the BioPlex 2200 ANA Screen analysis of 13 different analytes (dsDNA, centromere B, chromatin, Jo1, ribosomal P, RNP 68, RNP A, Scl‐70, Sm, SmPNP, SS‐A52, SS‐A60, SS‐B) is comparable (P < 0.252) to the ELISA ANA screening test. Like the ELISA, the BioPlex 2200 has a lower (P < 0.001) positive rate than IIF for the autoantibody screening.

Early Atherosclerosis in Rheumatoid Arthritis: Effects of Smoking on Thickness of the Carotid Artery Intima Media

Abstract: This study was designed to compare intima media thickness (IMT) of the carotid arteries among rheumatoid arthritis (RA) patients and controls and to determine whether disease‐associated characteristics, smoking, and other classic risk factors for atherosclerosis are associated with IMT values. IMT was measured in the carotid arteries of 101 RA patients and 75 control subjects. The IMT was evaluated in the common carotid (CC), carotid bifurcation (BI), and internal carotid (IC). Eight IMT values were calculated including four mean and four maximal values of CC, BI, IC, and carotid artery (C). The following data were obtained for every patient: age, sex, body mass index (BMI), presence of erosions, extra‐articular manifestations, rheumatoid factor, medications, hypertension, hypercholesterolemia, diabetes mellitus, smoking status, daily number of cigarettes, number of smoking years, family history of cardiovascular diseases (CVD), and erythrocyte sedimentation rate (ESR) levels. RA patients had significantly higher mean‐BI IMT than controls (1.02 mm vs. 0.89 mm; P < 0.01), higher incidence of increased mean‐BI IMT and max‐BI IMT, but lower incidence of increased max‐IC IMT than controls. Factors significantly associated with IMT in the controls were age, BMI, and hypertension, whereas factors significantly associated with IMT in RA patients were age and smoking status. Mean carotid IMT was associated with all characteristics related to smoking in RA patients. Current smokers had higher mean carotid IMT and internal carotid artery IMT than former smokers. RA is associated with higher carotid artery bifurcation IMT. The profile of factors associated with IMT values is different between RA patients and controls. Smoking is an important factor augmenting early atherosclerosis in RA patients.