Ragnar Alm

Identification of Immune Responses Against Aldehyde-Modified Peptide Sequences in ApoB Associated With Cardiovascular Disease

Atherosclerosis develops as a result of a chronic arterial inflammation and intimal fibrosis. The disease represents in many respects a vascular repair process activated in response to injury caused by toxic breakdown products of aggregated and oxidized lipoproteins. The initial response of the artery involves expression of adhesion molecules and recruitment of leukocytes. Degenerated lipoproteins are removed from the extracellular space by macrophages. If lipoproteins continue to accumulate, the inflammatory process becomes chronic and cytokines stimulate smooth muscle to migrate into the intima. These cells proliferate and form an atherosclerotic plaque. Plaque cell death and inflammation in response to oxidized lipids and other toxic factors may cause plaques to rupture.Objective—Atherosclerosis is associated with an immune response against oxidized LDL, which modulates the progression of the disease process. Methods and Results—Using a library of polypeptides covering the complete sequence of apoB-100, the only major protein of LDL, we have identified over 100 different human antibodies reacting against aldehyde-modified apoB-100 sequences. IgM antibody titer levels decreased with age and were associated with the intima-media thickness of the carotid artery in subjects younger than 60 years. There were also inverse associations between IgM levels and oxidized LDL in plasma. In prospective clinical studies, antibody levels against several aldehyde-modified apoB-100 sites were associated with cardiovascular disease in this age group. Whether this immune response is adaptive (protective) or maladaptive (causal) in atherosclerosis requires further investigation. Conclusions—We have characterized a large number of epitopes within the apoB-100 component of oxidized LDL that provoke an immune response in humans. These findings will make it possible to study the role of immune responses against specific sites in oxidized LDL and to determine whether these immune responses influence the risk for future cardiac events.

Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes

Aims/hypothesisOxidation of LDL in the arterial extracellular matrix is a key event in the development of atherosclerosis and autoantibodies against oxidised LDL antigens reflect disease severity and the risk of developing acute cardiovascular events. Since type 2 diabetes is associated with increased oxidative stress, we tested the hypothesis that autoantibodies against oxidised LDL antigens are biomarkers for vascular complications in diabetes.MethodsWe studied 497 patients with type 2 diabetes without clinical signs of coronary heart disease. Oxidised LDL autoantibodies were determined by ELISA detecting IgG and IgM specific for native and malondialdehyde (MDA)-modified apolipoprotein B-100 peptides p45 and p210. The severity of coronary disease was assessed as the coronary artery calcium score.ResultsPatients affected by retinopathy had significantly higher levels of IgG against MDA-p45 and MDA-p210. In contrast, high levels of autoantibodies against the corresponding native peptides were associated with less coronary calcification and a lower risk of progression of coronary disease.Conclusions/interpretationOur observations suggest that LDL oxidation is involved in the pathogenesis of diabetic retinopathy and that autoantibodies against apolipoprotein B peptides may act as biomarkers for both micro- and macrovascular complications in diabetes.

Association Between IgM Against an Aldehyde-Modified Peptide in Apolipoprotein B-100 and Progression of Carotid Disease

Background and Purpose— Autoantibodies against antigens in oxidized low-density lipoprotein are common in people; experimental studies suggest that these immune responses have a functional role in the disease process. The aim of this study was to evaluate the relationship between the immune response against one defined oxidized low-density lipoprotein antigen, the aldehyde-modified peptide corresponding amino acids 3136 and 3155 (MDA-p210) in apolipoprotein (apo) B-100, and progression of carotid intima media thickness (IMT). Methods— IgM and IgG against MDA-p210 were determined by enzyme-linked immunosorbent assay at baseline and after 12 months of treatment with placebo, metoprolol, fluvastatin, or metoprolol/fluvastatin in 751 individuals participating in the BCAPS. Carotid IMT was assessed by ultrasonography at baseline and after 18 and 36 months of treatment. Results— Antibody levels did not change in response to treatment, but high baseline MDA-p210 IgM levels were associated with a more rapid progression of carotid disease both at 18 (r=0.09, P<0.05) and 36 months (r=0.12, P<0.005). At 36 months, the difference in IMT progression rate per year between those with high MDA-p210 IgM levels and those with low was 0.011 mm (95% CI=0.005 to 0.018 mm, P<0.0001). Treatment with fluvastatin markedly decreased the progression of IMT among subjects with high but not with low MDA-p210 IgM levels. There was no association between MDA-p210 IgG and carotid IMT progression. Conclusions— IgM against the aldehyde-modified peptide corresponding amino acids 3136 and 3155 in apo B-100 is common in subjects with asymptomatic carotid disease, and high levels are associated with a more rapid progression of carotid IMT. The observation that the effect of fluvastatin was restricted to subjects with high MDA-p210 IgM levels may reflect the increased rate of disease progression in this group.

Patients with irritable bowel syndrome and dysmotility express antibodies against gonadotropin-releasing hormone in serum

Background  The etiology of irritable bowel syndrome (IBS) and dysmotility is in most cases unknown. Organic, pathognomonic changes have not been described. We have previously demonstrated sporadic expressions of antibodies against gonadotropin‐releasing hormone (GnRH) in serum from these patients. The aim of this study was to screen for the presence of GnRH antibodies in healthy subjects and patients with gastrointestinal (GI) diseases.

Prevalence and Clinical Significance of Gliadin Antibodies in Healthy Children and Adults

Coeliac disease (CD) is associated with the presence of gliadin antibodies (GA) (IgG and IgA), often used as a screening test for CD. Using a modified micro-enzyme-linked immunosorbent assay for GA, we studied the prevalence of GA in three healthy groups: children (mean age, 12 years), adult blood donors (mean age, 38 years), and healthy women (mean age, 57 years). We also studied the clinical characteristics of the blood donors. On the basis of findings in 27 untreated CD patients, cut-off levels of IgG and IgA antibody titres were chosen to yield a test with relatively low sensitivity (56%) but high specificity (100%). Analysis of IgM antibodies did not improve the sensitivity. Of the 384 12-year-old children, both IgG and IgA GA positivity was found in 15 (3.91%), a rate significantly greater than that in the blood donors (22 of 1537, 1.43%; p < 0.001) or in the middle-aged women (11 of 944, 1.17%; p < 0.0001). Of the 22 GA-positive healthy blood donors, 13 underwent small-bowel biopsy, but only 1 of the specimens manifested histologic changes compatible with CD. The other 12 had normal specimens, including a normal intraepithelial lymphocyte count. The estimated frequency of CD among the blood donors was thus 1 of 1500, a figure consistent with those previously published. We conclude that GA occur frequently in the Swedish population but that their prevalence decreases with increasing age. As a screening test for CD in healthy individuals, the GA titre is of poor predictive value.

The microheterogeneity of desialylated α1-antichymotrypsin: the occurrence of two amino-terminal isoforms, one lacking a His-Pro dipeptide

ACT (alpha 1-antichymotrypsin), a serine antiproteinase with specificity against neutrophil cathepsin G, is homologous with alpha 1-antitrypsin, plasminogen activator inhibitor and angiotensinogen, all with known amino-terminal microheterogeneity. Here we report that the two predominant isoforms of desialylated ACT obtained on isoelectric focusing correspond to a microheterogeneity at the amino terminus of ACT: one isoform (His-Pro-Asn-Ser-Pro-) and a two residues shorter isoform (Asn-Ser-Pro-). The relative occurrence of the two isoforms was comparable both in normal plasma, acute-phase plasma and plasma from subjects with heterozygous familial ACT deficiency. When desialylated ACT, isolated by affinity chromatography from ACT-deficient, normal or acute-phase plasma, was compared with regard to mass and charge microheterogeneity, we found no significant differences in either respect. Nor was the isoform pattern of desialylated plasma from patients with rheumatoid arthritis different. Although the occurrence of heterozygous familial ACT deficiency implies genotypic variation, isolated ACT from patients with the trait was not found to exhibit any phenotypic variation detectable by standard electrophoretic methods.

Organ cultures of human fetal hepatocytes in the study of extra- and intracellular α1-antitrypsin

The rate of synthesis of alpha 1-antitrypsin has been studied in organ cultures of fetal human liver. By de novo synthesis, alpha 1-antitrypsin of the same electrophoretic mobility and molecular size as plasma alpha 1-antitrypsin was produced. Synthetic rate was comparable to in vivo conditions and was suppressed by cycloheximide, colchicine and neuraminidase. By increasing alpha 1-antitrypsin levels in cultre medium, suppression of alpha 1-antitrypsin release from the intra-to the extracellular site was achieved, i.e., synthesis does not proceed autonomously. This suppression was preceded by a temporary enhancement of synthesis. Both effects were found to be independent of degree of sialylation of add-d alpha 1-antitrypsin. In contrast to alpha 1-antitrypsin released in tissue culture, the intracellular protein, as analyzed by crossed immunoelectrophoresis of Triton X-100 extracts from fetal liver, was found to occur partly as slowly moving peaks. Whether these peaks represent proforms or incompletely glycosylated precursors of export alpha 1-antitrypsin or complexes with proteases remains unsettled. A variety of other plasma proteins are released in organ cultures making the system suitable for study of factors regulating plasma protein synthesis.

Depletion of enteric gonadotropin-releasing hormone is found in a few patients suffering from severe gastrointestinal dysmotility.

Abstract Objective. Many patients, especially women, suffer from severe gastrointestinal pain and dysmotility for several years without being diagnosed. Depletion of gonadotropin-releasing hormone (GnRH) in the enteric nervous system (ENS) has been described in some patients. The aim of this study was to examine the expression of GnRH in ENS and antibodies against GnRH in serum, in a dysmotility patient cohort of southern Sweden. Materials and methods. All consecutive patients (n = 35) referred for laparoscopic full-thickness biopsy because of symptoms or signs of severe dysmotility between 1998 and 2009, or patients with a severe dysmotility disorder having had a bowel resection within the time frame, were considered for inclusion. In 22 cases, representative biopsy material containing ganglia was available, and these patients were included. Medical records were scrutinized. The expression of GnRH was determined by immunohistochemistry in bowel biopsies from these patients and in patients with carcinoma or diverticulosis without ENS histopathology. Antibodies against GnRH in serum were determined by ELISA in patients and controls. Results. 14 patients were diagnosed with enteric dysmotility (ED) and 8 with chronic intestinal pseudo-obstruction due to varying etiology. Immunostained biopsies showed expression of GnRH in the ENS. A reduced expression of GnRH-containing neurons was found in 5 patients, as well as antibodies against GnRH in serum. 3 of these patients had a history of in vitro fertilization (IVF) using GnRH analogs. Conclusions. A subgroup of patients with severe dysmotility had a reduced expression of GnRH-containing neurons in the ENS and expressed antibodies against GnRH in serum.

TAP1-Deficiency Does Not Alter Atherosclerosis Development in Apoe(-/-) Mice.

Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8+ T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8+ T cell population. We have recently reported an increased activation of CD8+ T cells in hypercholesterolemic Apoe−/− mice. Therefore, this study included TAP1-deficient Apoe−/− mice (Apoe−/−Tap1−/−) to test the atherogenicity of CD8+ T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8+ T cell numbers were low in Apoe−/−Tap1−/− mice in comparison to Apoe−/− mice, constituting ∼1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by en face Oil Red O staining of the aorta and cross-sections of the aortic root between Apoe−/−Tap1−/− and Apoe−/− mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3+ T cells in Apoe−/−Tap1−/− compared to Apoe−/− mice. The CD3+CD4+ T cell fraction was increased in Apoe−/−Tap1−/− mice, suggesting a compensation for the decreased CD8+ T cell population. Interestingly, the fraction of CD8+ effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development. In conclusion, Apoe−/−Tap1−/− mice develop atherosclerosis equal to Apoe−/− mice, indicating a minor role for CD8+ T cells and TAP1-dependent antigen presentation in the disease process.

Circulating CD40+ and CD86+ B Cell Subsets Demonstrate Opposing Associations With Risk of Stroke

Objective— Accumulating evidence shows that immune cells play an important role in atherosclerosis. Most attention has focused on the role of different T cell subsets, whereas the possible involvement of B cells has been less studied. In this study, we assessed the association of 2 different B cell subsets, CD19+CD40+ and CD19+CD86+ B cells, with risk for development of acute cardiovascular events. Approach and Results— The prospective study included 700 subjects randomly selected from the cardiovascular cohort of the Malmö Diet and Cancer study. Mononuclear leukocytes, stored at –140○C at the baseline investigation in 1991–1994, were thawed and B cell subsets analyzed by flow cytometry. Cytokine release from CD3/CD28-stimulated mononuclear leukocytes was measured with multiplex ELISA. Baseline carotid intima-media thickness and stenosis were assessed by ultrasonography, and clinical events were monitored through validated national registers during a median/mean follow-up time of 15 years. The subjects in the highest tertile of CD19+CD40+ B cells had a significantly lower risk of incident stroke after adjustment for other risk factors. In contrast, CD19+CD86+ B cells were associated with higher risk for development of a stroke event and increased release of proinflammatory cytokines from mononuclear leukocytes. Conclusions— These observations provide evidence for an involvement of B cells in the incidence of stroke and suggest that both pathogenic and protective B cell subsets exist.