Anti-idiotypes to oxidized LDL antibodies in intravenous immunoglobulin preparations--possible immunomodulation of atherosclerosis

OBJECTIVE: The aim of this study was to examine whether intravenous immunoglobulin (IVIg) preparations contain anti-oxLDL and anti-anti-oxLDL antibodies. BACKGROUND: Oxidized low-density lipoprotein (oxLDL) is one of the major players in atherogenesis. IVIg can reduce atherosclerosis in experimental animal models. METHODS: Six commercial IVIg preparations were tested for the presence of anti-oxLDL antibodies by EIA. Inhibition studies were performed with the different IVIg preparations and IgGs purified from a pool of sera from patients with high anti-oxLDL antibody levels. Absorption assays were carried out to evaluate the presence of anti-idiotypes against anti-oxLDL antibodies in IVIg preparations. RESULTS: IVIg preparations tested had various degrees of reactivity towards oxLDL. Absorption experiments suggested that the reactivity was specific because it could be effectively absorbed by oxLDL and not by an irrelevant antigen PPD. The reactivity was smaller than that observed with the IgG from the pool with high anti-oxLDL antibody levels. Inhibition studies with IVIg demonstrated 20-45% inhibition of anti-oxLDL binding to oxLDL, compared to 76% inhibition by the pool with high anti-oxLDL levels. To investigate the presence of anti-idiotypes against anti-oxLDL antibodies within IVIg, F(ab')2 fragments of IVIg IgG were used to absorb IgG F(ab')2 fragments from the pool of sera with high anti-oxLDL levels. The decreased binding to oxLDL of the absorbed supernatants shows that IgG F(ab')2 fragments of the IVIg preparations had high inhibitory capacities ranging from 65 to 90%. CONCLUSIONS: IVIg preparations contain both anti-oxLDL and anti-anti-oxLDL activity. This finding may explain the immunomodulating effect of IVIg in atherosclerosis.     

The influence of different cultivating conditions on polymorphonuclear leukocyte apoptotic processes in vitro,I: the morphological characteristics of PMN spontaneous apoptosis

Polymorphonuclear leukocyte (PMN) populations incubated in vitro with normal human serum are save-regulated systems of spontaneous apoptosis. Light microscopy (LM), transmission (TEM), and scanning (SEM) electron microscopes were used for the evalution of PMN apoptopic alteration. Twelve-hour PMN populations were represented by optimal number of normal and different apoptotic forms. Their ultrastructural analysis showed that on this background, 3 apoptotic cell lines (code named "first," "second," and "third") were predominated. The following characteristics were featured: "first"--vacuolization of same organelles, release of their content outside, increase of general cytoplasmic density, nuclear filling with condensed chromatin, and formation of PMNs mainly into small, round, dense forms; "second"--involvement of micronuclei or nuclei in apoptosis, their displacement to the cytoplasmic membrane and separation from the cells, and cytoplasm had numerous intact granules almost until the completion of apoptosis; "third"--synchronous apoptotic process of the nuclei and cytoplasm, moderate electronic density of cytoplasm, and granular translocation to the cell surface. Secondary necrosis was completed mainly in the apoptotic process of the "second" and "third" lines. SEM surfaces confirmed the results of TEM. This research showed that neutrophil spontaneous apoptosis is a complicated process. The 3 apoptotic cell lines reflect different pathways characteristic for the studied systems under certain conditions of cultivation.     

A novel system to diagnose cutaneous adverse drug reactions employing the cellscan--comparison with histamine releasing test and Inf-gamma Releasing Test

Background: There are several mechanisms to describe allergic drug reactions yet the methods to diagnose them are limited. Objective: To compare several conventional clinical and laboratory methods to diagnose skin reactions to drugs to a new method of diagnosing drug reactions by the CellScan system. Methods: The study entailed 21 patients who were diagnosed as suffering from drug eruptions, and 105 healthy controls with no history of drug allergy. The drugs were classified into two groups according to suspicion of causing drug allergy: high and low. Most of the patients were on more than one drug, leading to 41 patient-drug interactions (assays). Histamine releasing test (HRT), interferon (INF)-γ releasing test and CellScan examination were performed on lymphocytes of the patients and controls. Results: The HRT was interpreted as positive in 9 out of 18 (50%) patients and in 13 out of 35 (37%) assays. Based on the INF-γ releasing test, positive results were observed in 16 out of 21 (76%) patients and in 24 out of 41 (59%) assays. In the CellScan test (CST), positive results were observed in 17 out of 21 (81%) patients and in 29 out of 41 (71%) assays. The rate of identifying the drug for eruption in the high suspicion level drugs was 9 out of 22 (41%) assays in the HRT, 20 out of 24 (83%) assays in the INF-γ releasing test, and 21 out of 24 (87%) studies with the CellScan method. The rate of determining of the drug that caused the eruption in the low suspicion level drugs was 4 out of 13 (31%) in the HRT, 4 out of 17 (24%) assays in the INF-γ releasing test, and 8 out of 17 (47%) analyses in the CST. When examined in the CellScan, 99 out of 105 (94%) controls were interpreted as negative. Conclusion: This preliminary study indicates that the CellScan seems to be an easy and promising method for the detection of drugs responsible for adverse skin reactions. In contrast to the HRT and to the Interferon-γ secretion test, the CellScan method is characterized by its ability to track and monitor the reaction of individual cells. By measuring the kinetic parameters of selected cells before and after adding the suspected drug, we were able to identify the culprit drug. The CellScan method had the highest sensitivity, and the interferon-γ secretion test had the highest specificity for detection of the culprit drug. In contrast, the analysis of 105 normal control sera disclosed a high specificity of 94% for the CellScan method.     

Induction of biologically active antineutrophil cytoplasmic antibodies by immunization with human apoptotic polymorphonuclear leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.     

The Influence of Different Cultivating Conditions on Polymorphonuclear Leukocyte Apoptotic Process In Vitro,II: Ultrastructural Characteristics of PMN Populations Incubated with Proteinase 3 Anti-neutrophil Autoantibodies

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27:23–32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40 min and 12 h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named “first,” “second,” and “third.” The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the “first” line is characterized by intensification and modification of activation; the “second” by vacuolized cell forms; and the “third” by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.     

Anti-idiotypes to Oxidized LDL Antibodies in Intravenous Immunoglobulin Preparations--Possible Immunomodulation of Atherosclerosis

Objective : The aim of this study was to examine whether intravenous immunoglobulin (IVIg) preparations contain anti-oxLDL and anti-anti-oxLDL antibodies. Background : Oxidized low-density lipoprotein (oxLDL) is one of the major players in atherogenesis. IVIg can reduce atherosclerosis in experimental animal models. Methods : Six commercial IVIg preparations were tested for the presence of anti-oxLDL antibodies by EIA. Inhibition studies were performed with the different IVIg preparations and IgGs purified from a pool of sera from patients with high anti-oxLDL antibody levels. Absorption assays were carried out to evaluate the presence of anti-idiotypes against anti-oxLDL antibodies in IVIg preparations. Results : IVIg preparations tested had various degrees of reactivity towards oxLDL. Absorption experiments suggested that the reactivity was specific because it could be effectively absorbed by oxLDL and not by an irrelevant antigen PPD. The reactivity was smaller than that observed with the IgG from the pool with high anti-oxLDL antibody levels. Inhibition studies with IVIg demonstrated 20-45% inhibition of anti-oxLDL binding to oxLDL, compared to 76% inhibition by the pool with high anti-oxLDL levels. To investigate the presence of anti-idiotypes against anti-oxLDL antibodies within IVIg, F(ab') 2 fragments of IVIg IgG were used to absorb IgG F(ab') 2 fragments from the pool of sera with high anti-oxLDL levels. The decreased binding to oxLDL of the absorbed supernatants shows that IgG F(ab') 2 fragments of the IVIg preparations had high inhibitory capacities ranging from 65 to 90%. Conclusions : IVIg preparations contain both anti-oxLDL and anti-anti-oxLDL activity. This finding may explain the immunomodulating effect of IVIg in atherosclerosis.     

The prediction of coronary atherosclerosis employing artificial neural networks

BACKGROUND: Atherosclerosis is a complex histopathologic process that is analogous to chronic inflammatory conditions. Several factors have been shown to correlate with the extent of atherosclerosis. Whereas hypertension, obesity, hyperlipidemia, diabetes, smoking, and family history are all well documented, recent literature points to additional associated factors. Thus, antibodies to oxidized low-density lipoprotein (oxLDL), cytomegalovirus (CMV), Chlamydia pneumonia, Helicobacter pylori, as well as homocysteine and C-reactive protein (CRP) levels have all been implicated as independent markers of accelerated atherosclerosis. HYPOTHESIS: In the current study we attempted to formulate a system by which to predict the extent of coronary atherosclerosis as assessed by angiographic vessel occlusion. METHODS: The 81 patients were categorized as having single-, double-, triple-, or no vessel involvement. The clinical data concerning the "classic" risk factors were obtained from clinical records, and sera were drawn from the patients for determination of the various parameters that are thought to be associated with atherosclerosis. RESULTS: Using four artificial neural networks, we have found the most effective parameters predictive of coronary vessel involvement were (in decreasing order of importance) antibodies to oxLDL, to cardiolipin, to CMV, to Chlamydia pneumonia, and to beta 2-glycoprotein I (beta 2GPI). Although important in the prediction of vessel occlusion, hyperlipidemia, hypertension, CRP levels, and diabetes were less accurate. CONCLUSION: The results of the current study, if reproduced in a larger population, may establish an integrated system based on the creation of artificial neural networks by which to predict the extent of atherosclerosis in a given subject fairly and noninvasively.     

Intravenous gamma globulin inhibits the production of matrix metalloproteinase-9 in macrophages

BACKGROUND: Degradation of the extracellular matrix (ECM) is essential for progression and metastasis of cancer cells. The ECM-degrading enzymes, matrix metalloproteinases (MMPs), are produced mainly by intratumor monocytes/macrophages. MMPs, particularly MMP-9, are reported to be of crucial significance for both growth and tumor invasiveness. Inhibition of the expression of MMP-9 may prevent tumor development. High-dose intravenous gamma globulins (IVIG) effectively inhibit metastatic spread of tumors in mice and humans and a variety of mechanisms have been suggested to explain this effect. METHODS: We studied the effect of purified IVIG on MMP-9 secretion and mRNA expression by in vitro differentiated human monocytic cells (cell lines and peripheral blood monocytes). Zymography was employed to measure gelatinase secretion and Northern blot analysis was used to detect mRNA expression. Involvement of F(ab)(2) and Fc components in IVIG activity was also evaluated. RESULTS: IVIG dose dependently and significantly reduced the amount of secreted MMP-9 and its mRNA expression. F(ab)(2), but not Fc fragments, led to suppressed MMP-9 activity. However, competitive experiments demonstrated that Fc, but not F(ab)(2) fragments, reversed the IVIG-induced inhibitory effects. CONCLUSIONS: These results suggest that the whole IgG molecule may be needed for pertinent IVIG-induced MMP-9 down-regulation. This study points to an additional new mechanism whereby IVIG may play a beneficial role in the prevention of tumor spread in humans.     

Prevention of tumor spread by matrix metalloproteinase-9 inhibition: old drugs, new concept

In the following review we will discuss some familiar drugs with potential use as adjuvant anti-neoplastic agents. We will highlight their unfamiliar property of being able to inhibit matrix metalloproteinase-9 activity, which has recently been proven to play a key role in cancer spread. These drugs' high-safety profile may allow clinicians to construct new anti-cancer protocols that may prove to be less toxic alternatives to those commonly used.     

Anti-endothelial cell antibodies in patients with coronary atherosclerosis

Anti-endothelial cell antibodies (AECA) have been shown to possess endothelial cell activation properties and to harbor pathogenic potential in experimental animal models of autoimmune systemic disorders. Atherosclerosis is a form of an inflammatory condition in which the immune system has been shown to be involved. The aim of the present study was to assess the presence of AECA in patients with coronary atherosclerosis. A total of 134 patients admitted for chest pain of suspected anginal origin were evaluated for coronary artery atherosclerosis by angiography. Sera were drawn prior to the procedure for the determination of AECA employing cyto-ELISA. AECA positive sera were further evaluated for its ability to promote in vitro E-selectin expression by HUVEC using a cell-based ELISA. Patients with no coronary artery involvement had levels of AECA that did not differ from those obtained for patients with confirmed coronary atherosclerosis (one, two or three vessel disease). Furthermore, AECA positive sera from patients, with or without coronary atherosclerosis displayed similar capacity of inducing E-selectin expression by endothelial cells. AECA may not stand as an optimal mean of discriminating atherosclerotic from non-atherosclerotic patients. The ability of AECA to activate endothelial cells is also not unique to patients with atherosclerosis and is evident also in age-matched control subjects.