G. Virella

Isolation and Characterization of Human Antioxidized LDL Autoantibodies

Autoantibodies to oxidized LDL have been reported in normal subjects and in patients with arteriosclerosis, but their possible pathogenic role is not yet well defined. One important problem is the existence of contradictory data reported by different groups concerning the associations between antioxidized LDL autoantibodies and the presence or progression of arteriosclerotic lesions. Such contradictions led us to decide to isolate and characterize antioxidized LDL antibodies by affinity chromatography with the use of oxidized LDL cross-linked to Sepharose. Antioxidized LDL antibodies were isolated from selected serum samples obtained from eight subjects. Seven of them (six patients and one control subject) had high levels of antioxidized LDL antibody during screening. The other subject, a healthy volunteer, had a low level of antibody. All purified antibodies contained IgG (of subclasses 1 and 3) as the predominant isotype and were primarily specific for oxidized LDL but showed some cross-reactivity with malondialdehyde-modified LDL and native LDL. Two of the purified antibodies cross-reacted with cardiolipin. We determined average dissociation constants for the antioxidized LDL antibodies purified from five individuals, which varied between 2.4 x 10(-7) and 7.5 x 10(-7) mol/L, whereas the average dissociation constant of rabbit hyperimmune anti-LDL antibody was determined to be 2.7 x 10(-8) mol/L. In conclusion, we have purified human autoantibodies reactive with oxidized LDL that appear to be predominantly of moderate-to-low affinity and of variable cross-reactivity. The predominance of IgG1 and IgG3 antibodies is significant from the standpoint of potential pathogenicity, since these two subclasses activate the classic complement pathway system and have the highest binding affinities for Fc gamma receptors on phagocytic cells.

Isolation of soluble immune complexes by affinity chromatography using staphylogoccal protein A-Sepharose as substrate

This report describes a technique for the general isolation of immune complexes, based on a combination of gel filtration and affinity chromatography. The first step is the preparation of a globulin-enriched fraction by precipitation with ammonium sulfate at 50% saturation, or of an immune-complex-enriched fraction by precipitation with 5% polyethylene glycol 6000. The enriched fraction is then subfractionated by gel filtration in Ultrogel AcA 34. The immune complexes elute close to the void volume in the macroglobulin peak, separated from monomeric IgG molecules. This peak (sometimes subdivided into two fractions) is then submitted to affinity chromatography on a protein A--Sepharose cooumn. Most immune complexes contain IgG molecules and therefore bind to the column. Almost no protein is bound when normal serum is fractionated according to this method, and no immunoglobulins are detectable in the acid-eluted fraction from the protein A--Sepharose column. In two patients with soluble immune complexes in their sera we eluted immunoglobulin-containing fractions from the column; in one, these fractions had high rheumatoid factor titers; and in the second, with a clinical diagnosis of systemic lupus erythematosus, a similar fraction contained RNA.

Levels of Oxidized LDL and Advanced Glycation End Products–Modified LDL in Circulating Immune Complexes Are Strongly Associated With Increased Levels of Carotid Intima-Media Thickness and Its Progression in Type 1 Diabetes

OBJECTIVE High cholesterol levels in circulating immune complexes (IC), surrogate markers of modified LDL, are associated with increased carotid intima-media thickness (IMT) and cardiovascular events in type 1 diabetes. Different modifications of LDL are involved in IC formation, but which of these are predictive of vascular events is not known. Therefore, we measured oxidized LDL (oxLDL), advanced glycation end products–modified LDL (AGE-LDL), and malondialdehyde-modified LDL (MDA-LDL) in IC and determined their relationship with increased carotid IMT and compared the strength of the association with that observed with conventional risk factors. RESEARCH DESIGN AND METHODS Levels of oxLDL, AGE-LDL, and MDA-LDL were measured in circulating IC isolated from sera of 479 patients of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort, collected at baseline. Internal and common carotid IMT were measured 8 and 14 years later by DCCT/EDIC. RESULTS OxLDL, AGE-LDL, and MDA-LDL levels in circulating IC were significantly correlated with diabetes duration, BMI, and lipid and blood pressure, but not with age. Multivariate logistic regression models indicated that individuals in the highest versus lowest quartile of oxLDL and AGE-LDL in IC had a 6.11-fold [confidence interval (CI) 2.51–14.8] and a 6.4-fold (CI 2.53–16.2) increase in the odds of having high carotid IMT, respectively, after adjusting for conventional risk factors. Parallel analyses resulted in odds ratios of 2.62 (CI 1.24, 5.55) for LDL-C, 1.45 (CI 0.69, 3.03) for diastolic blood pressure, and 2.33 (CI 1.09, 4.99) for A1C. CONCLUSIONS OxLDL and AGE-LDL in circulating IC were significantly associated with progression and increased levels of carotid IMT in type 1 diabetes.

X-Linked Immunodeficiency with Increased IgM: Clinical, Ethnic, and Immunologic Heterogeneity

Summary: Two cases of immunodeficiency with increased IgM are reported. Patient 1 was a black male 3.5 years old who had recurrent pyogenic infections, failure to thrive, oral thrush, and systemic cryptococcal infection. Patient 2 was a 9-year-old white female who had recurrent cervical abscesses. Serum immunoglobulin determinations by radial immunodiffusion in both patients showed marked depression of IgG and IgA and marked elevation of IgM. A low molecular weight circulating monomeric IgM was demonstrated by immunoelectrophoresis and gel filtration in the second patient; this was not present in the first case. In vitro impairment of cellular immunity was observed in both patients. Administration of dialyzable leukocyte extracts (transfer factor) led to improvement of cell-mediated immunity in patient 1. The etiology of this syndrome apparently has several different genetic bases. These patients demonstrate heterogeneity in genetic, ethnic, immunologic, and other features of the syndrome.Speculation: The cause of hyper-IgM syndrome with depressed synthesis of IgA and IgG immunoglobulins is unknown. Deficiency in the function of T cells1 or a subpopulation thereof could have a causal relation to this syndrome, since there is increasing evidence that normal cellular immunity is necessary for the IgM-to-IgG “switch” in immunoglobulin production; whether it is also necessary for the formation of normal polymeric IgM from monomeric IgM is unknown, but this possibility merits speculation. In the present study, the occurrence of monomeric IgM in one patient but not the other, along with the differences in clinical manifestations, raises the possibility that the presence of monomeric IgM may be an index of a distinct subcategory of this syndrome with certain clinical features present only when the monomeric molecule is found. Our two patients, and all eight previously reported patients in whom isohemagglutinins were studied, had isohemagglutinins for A and B cells (i.e., were blood type O). In addition, two other hyper-IgM patients seen by us subsequently have been found to be blood type O (unpublished observation). If this phenomenon is universal, it would suggest a very close association between the blood group O antigen and this syndrome.

Urinary high density lipoprotein in minimal change glomerular disease and chronic glomerulopathies.

Serum lipids and lipoproteins and urinary apolipoprotein A (Apo A) were determined in two groups of patients. One group consisted of 11 children (ages ranging from 4 to 14 years) with minimal change glomerular disease. The other group consisted of 13 patients, eight less than 19 years old five adults, with different types of chronic glomerulopathy. Elimination of urinary lysozyme was a feature of chronic glomerulopathies, and creatinine clearances were also significantly lower in this group. Patients with chronic glomerulopathies had significantly lower HDL cholesterol and Apo A concentrations in their sera. In contrast, urinary Apo A concentrations were significantly higher in patients with chronic glomerulopathies, who also showed significantly lower urinary protein selectivities. Lipoprotein electrophoresis of urines containing Apo A showed distinct high-density lipoprotein (HDL) fractions, suggesting that HDL is eliminated in the urine as a result of increased glomerular permeability. This is also supported by a correlation coefficient of 0.77 between the selectivity indices and the ratio of urinary Apo A to total proteinuria. The determination of urinary Apo A appears to give valuable diagnostic information in patients with glomerular disease. According to our results the absence of urinary Apo A is very suggestive of minimal change glomerular disease.

New method for obtaining IgA-specific protease.

A simple method for obtaining an active preparation of IgA-specific protease from a bacterial source is presented. In this method Streptococcus sanguis was inoculated onto the surface of a dialysis membrane on nutrient agar. Following growth, the membrane was removed from the agar surface and washed in a small volume of buffer. A solution with protease activity against IgA1 monoclonal proteins was obtained by clarification of the wash and appeared to be similar to enzyme preparations obtained by other methods.

Immune Mechanisms in the Pathogenesis of Atherosclerosis

It is generally accepted that arteriosclerosis is a multifactorial disease and that several risk factors contribute to its development. However the correlation between the development of arteriosclerosis and the presence of any of the known risk factors or associations of risk factors is not perfect. Furthermore, the precise mechanism by which each risk factor contributes to the pathogenesis of arteriosclerosis is not well understood. Thus, there has been a persistent development of new thoughts and theories concerning risk factors and their relative pathogenic roles. In the past decade an upsurge of interest in the role of immune mechanisms in the development of arteriosclerosis has emerged. In the present review we will analyzed data suggesting that immunological factors may contribute, directly or indirectly, to the sequence of pathological events leading to the development of arteriosclerosis.

Differential sensitivity of IgA proteins of different subclasses and allotypes to reduction of disulfide bonds and digestion by streptococcal protease

Abstract Twenty purified IgA proteins were typed for subclass and allotype 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 contamination of an IgAl protein with IgA2 A2m (1) molecules or, more likely, to the existence of an IgA2 A2m (1) protein with disulfide bonds joining the heavy and light chains. The second case was apparently a mistyping, since the protein behaved as IgA1 by all immunochemical criteria and the reaction with anti-IgA2 A2m (1) was weak. Upon incomplete reduction of interchain bonds, most of the IgAl proteins and all the IgA2 A2m (1) proteins released predominantly H2 subunits; but in a small number of IgA1 proteins, release of HL subunits was equal to or greater than the release of H2, suggesting that additional serotypes of IgA remain to be defined.

Asynchronous development of two monoclonal proteins (igm λ and γ1 chains) in a patient with abdominal lymphoma

An IgM paraprotein was detected in a patient with abdominal lymphoma. Due to some doubts about the malignant nature of the situation, no cytostatic drugs were administered. When seen two years later the patient had considerably deteriorated in clinical status, with evidence of extra‐abdominal involvement. At this time he presented a second paraprotein of low of molecular weight (74,000 to 80,000), consisting of two covalently linked γ1‐chain fragments.

Urinary lysozyme phenotypes in monocytic leukemia

A method of two-dimensional electrophoresis has been devised to allow the study of the electrophoretic mobility of urinary lysozyme. Three different phenotypes have been defined in a study of thirteen purified lysozymes obtained from different patients with monocytic leukemia.