Anticardiolipin and anti-beta2-glycoprotein I antibodies in Behcet's disease.

To investigate prevalence of anticardiolipin antibodies (aCL) in patients with Behcet''s disease (BD) and to determine whether they are related to anti-beta2-glycoprotein I antibodies (aGPI), we measured aCL and aGPI in 47 patients of BD and 14 patients of systemic lupus erythematosus (SLE). The levels of aCL and aGPI were determined by conventional enzyme immunoassay for both IgG and IgM classes. Twelve (25.5%) patients with BD were positive for IgG or IgM aCL and no patient was positive for aGPI. Eleven (78.6%) patients with SLE were also positive for aCL and among them, 8 (72.7%) patients were positive for aGPI. Positive IgG aCL patients with BD showed lower level of IgG aCL than those with SLE (15.7+/-7.3 vs 34.1+/-16.0 GPL, p<0.05). There was no relation between the presence of aCL in BD and either dinical activity or clinical features. In the patients with BD, aCL are found but it would not be associated with aGPI as they are in patients with SLE. In patients with BD, aCL seem to be authentic aCL unlike those in patients with SLE and may not be related with vascular complications in BD.     

The orientation of beta2GPI on the plate is important for the binding of anti-beta2GPI autoantibodies by ELISA

(Beta2-glycoprotein I (beta2GPI) is a plasma protein that plays an important role in the antigenic specificity of antiphospholipid autoantibodies (aPL). These antibodies are associated with an increased risk for thrombosis and recurrent foetal loss in humans. Crystallographic analysis of beta2GPI showed that its five complement control protein (CCP) or 'sushi' domains are arranged in an elongated, fish-hook shape; yet the domain-specific location of epitopes recognized by these autoantibodies has remained the subject of considerable controversy. Investigators have used different forms of recombinant beta2GPI and different ELISA methods to obtain conflicting results. One group mapped autoimmune epitopes to domain I using deletion mutants of beta2GPI in a competitive inhibition ELISA on NUNC Maxisorp microplates. Another group mapped epitopes to domain IV using beta2GPI with mutations in domain IV in a direct binding ELISA on polyoxygenated microplates. In an effort to resolve these discrepancies, a collaboration between the groups compared wildtype beta2GPI with domain IV mutants in both types of ELISA. Autoantibodies bound very poorly to domain IV mutants coated on polyoxygenated plates, yet they bound very well to the same mutants coated on NUNC Maxisorp plates. The amount of protein adsorbed on to both types of plates was similar. In the competitive inhibition ELISA, no difference could be detected between wildtype beta2GPI and domain IV mutants. These results strongly suggest that the orientation of beta2GPI on the microplate, and not necessarily the lateral density, plays the predominant role in the binding of autoantibodies.     

Anti-cardiolipin and anti-beta 2-glycoprotein I antibodies in celiac disease

Aims

To determine the frequency of anti-cardiolipin (aCL) and anti-β2-glycoprotein I antibodies (aβ2GPI) in celiac disease (CD) patients.

Patients and methods

Sixty-three untreated CD patients and 40 healthy blood donors (HBD) were studied. IgG, IgA and IgM aCL and aβ2GPI were detected by Elisa.

Results

The frequency of antiphospholipid antibodies (aPL) (aCL and/or aβ2GPI) was significantly higher in CD patients (12 out of 63) than in HBD (two out of 40) (19% vs 5%, P = 0.04). Six CD patients out of 63 (9.5%) and one HBD out of 40 (2.5%) had aCL. Ten CD patients (15.9%) and two HBD (5%) had aβ2GPI. Only aβ2GPI-IgA was significantly more frequent in CD patients than in HBD (14.3% vs 2.5%, P = 0.048). In CD patients, aβ2GPI-IgA (nine out of 63) was significantly more frequent (14.3%) than aβ2GPI-IgG (1.6%) and IgM (1.6%) (P = 0.008). In CD patients, the frequency of aCL-IgA and IgM was 6.3% (four out of 63) and aCL-IgG were not detected. Simultaneous presence of positive antibodies was found in four CD patients: one patient had four aPL, one had three aPL and two had two aPL. The four patients who had aCL-IgA had also aβ2GPI-IgA and three of them had a titer higher than 50 units. Among nine patients with aβ2GPI-IgA, four had a titer higher than 100 units. The highest titers were found in adults.

Conclusions

aPL and particularly aβ2GPI-IgA are frequent in CD. The significance of these antibodies has to be determined.     

Use of the ELISA to screen for anti-thymocyte and anti-beta 2-microglobulin antibodies in leprosy and SLE.

A report is given of the use of the enzyme-linked immunosorbent assay to measure antibody to preparations of human thymocyte membranes (HTMA) and to beta 2-microglobulin. The assay described is simple and rapid, and requires only small quantities of an easily stored membrane preparation. The advantages of this technique over conventional methods involving cytotoxicity are discussed. Raised levels of IgM antibody to beta 2-microglobulin were detected in sera from SLE patients. Raised levels of IgG and IgM antibody to HTMA were found in sera from most active lepromatous cases. Two of eight sera from SLE patients showed raised IgG anti-HMTA, but not raised IgM. An attempt was made to study the subclass of the IgG antibodies found, but when checked against purified human IgG myeloma proteins, the available anti-subclass sera were found to lack the necessary degree of specificity in this assay.     

Clinical significance of anticardiolipin and anti-beta2-glycoprotein I antibodies

BACKGROUND: Anticardiolipin (aCl) and anti-beta2-glycoprotein I (anti-beta2-gpI) antibodies are autoantibodies associated with the antiphospholipid syndrome (APS), which is characterized by both arterial and venous thrombosis and miscarriages. The scope of this study was to explore the clinical characteristics of patients with aCl and anti-beta2-gpI antibodies. METHODS: ACl were tested in 3,600 consecutive sera in our laboratory between January 1999 and June 2001. The clinical diagnosis and prevalence of thrombosis and pregnancy morbidity were retrospectively reviewed in aCl-positive patients. Furthermore, the frequency of anti-beta2-gpI antibodies, lupus anticoagulant (LA), prolonged activated partial thromboplastin time (aPTT), and thrombocytopenia were investigated in aCl-positive patients. RESULTS: 147 aCl-positive patients, 110 women and 37 men with a mean age of 41 years (range 7.8-82.5), were identified. 42 (28.6%) aCl-positive patients fulfilled the criteria for APS which was secondary to a connective tissue disorder in 8 patients. The frequency of anti-beta2-gpI antibodies and LA, prolonged aPTT, and thrombocytopenia in aCl-positive patients was 23.8, 27.2, 25.7 and 9.2%, respectively. The presence of both aCl and anti-beta2-gpI antibodies was strongly associated with clinical symptoms of APS (p = 0.007) compared to p = 0.008 for LA. CONCLUSION: Our data suggest that assessment of anti-beta2-gpI antibodies in addition to aCl is a valuable diagnostic tool in the workup of patients with APS.     

High prevalence of anti-beta2 glycoprotein I antibodies in patients with ischemic heart disease.

Ischemic cardiac manifestations have been reported in a various percentage of patients with anti-phospholipid antibodies. As concerns the relationship between anti-beta2 glycoprotein I antibodies (anti-beta2-GPI) and ischemic heart disease (IHD), it was investigated in only one coronary primary prevention study. We investigated the prevalence of anti-beta2-GPI in a well characterized group of patients with different clinical manifestation of IHD. Sera from 37 patients (mean age 62.7 +/- 9.9) with IHD (20 with unstable angina-UA and 17 with effort angina-EA) and from 40 healthy subjects, matched for age and sex, were tested for the presence of IgG and IgM anti-beta2-GPI using an ELISA technique. Eleven/37 patients (29.7%) resulted positive for anti-beta2-GPI. A positivity for IgG anti-beta2-GPI was found in 10 patients, 1 patient was positive for IgM and 1 for both isotypes. The prevalence of anti-beta2-GPI in the control group resulted significantly lower (2.5%; p < 0.005) than in patients with IHD. Positivity for anti-beta2-GPI was found in 9/20 (45%) patients with UA and only in 2/17 patients (11.8%) with EA (p = 0.0365). IgG anti-beta2-GPI levels (median 7.7U/ml, range 2.6-24.1) were significantly higher in patients with UA compared to patients with EA (median 4.6 U/ml, range 2.3-11.5; p = 0.02) and controls (median 3.15 U/ml, range 2.3-9.0; p < 0.0001); also IgM levels resulted higher in patients with unstable angina. A positivity for anti-beta2-GPI was observed in 4/13 patients (30.8%) with a previous myocardial infarction (MI) and in 7/24 (29.2%) patients without a previous MI. Our findings suggest that anti-beta2-GPI could represent an expression of the T-cell activation detectable in patients with unstable angina. The lack of a significant difference in the prevalence of these antibodies in patients with or without a previous MI suggests that anti-beta2-GPI are not induced by tissue necrosis.     

Myocardial response to infarction in the rat. Morphometric measurement of infarct size and myocyte cellular hypertrophy.

For determination of the effects of myocardial infarction on the recovery potential of muscle mass in the surviving tissue, ligation of the left coronary artery was performed in 3-month-old rats, and the infarcted ventricles were analyzed morphometrically a month after surgery. Comparisons were made with 4-month-old control rats that underwent sham operations and with 3-month-old control rats that were not operated upon for evaluation of the magnitude of infarct size and discrimination of the relative contribution of tissue growth that occurred in the surviving myocardium solely as a result of the change in age, from 3 to 4 months (postoperative tissue growth, or POTG), from the additional growth induced by infarction (hypertrophic growth, or HG). Coronary occlusion induced a 276-cu mm loss of ventricular tissue volume that corresponded to 43% of the total left ventricular mass, 648 cu mm. Over a 30-day period the remaining 372 cu mm of viable tissue expanded by 90% with an overall volume gain of 334 cu mm. This tissue augmentation consisted of 20% POTG, 67 cu mm, and 80% HG, 267 cu mm. Total myocyte volume increased 89%, from 302 cu mm to 571 cu mm, and average myocyte cell volume per nucleus increased 92%, from 16,500 cu mu to 31,600 cu mu. The expansion of the myocyte mass was the result of a 21% POTG and a 79% HG. Corresponding values for the myocyte population were 19% and 81%.     

Relationship of anti-beta 2-microglobulin antibodies to T11 and T-cell activation in systemic lupus erythematosus

Monoclonal anti-beta 2-microglobulin (beta 2m) inhibited in a specific, dose-dependent fashion both in vitro tetanus toxoid-induced human-T-cell proliferation and sheep erythrocyte (E)-rosette formation, a function of the 50 kDa T11 molecule. In these respects, anti-beta 2m exhibited effects similar to those of sera from patients with SLE. Although 10 of 16 SLE sera contained antibody to beta 2m in monoclonal rosette inhibition assays, the presence of antibody of this specificity contributed only partially to the capacity of SLE serum to inhibit E-rosette formation or the T-cell response to tetanus toxoid. Removal of anti-beta 2m from SLE serum by solid phase absorption with beta 2m-Sepharose 4B reduced inhibition of the tetanus toxoid response and E-rosette formation in certain cases, but to a lesser extent than that observed following absorption with T-cell blasts, which completely eliminated inhibitory activity. Neither SLE antilymphocyte antibodies (including anti-beta 2m) nor heterologous anti-beta 2m were directed to the E receptor binding site (T11(1) epitope), as indicated by failure to inhibit OKT11 monoclonal antibody rosette formation or to reduce the relative intensity of OKT11 immunofluorescent staining. These data suggest an interesting functional relationship between beta 2m, the E receptor, and T-cell activation. While anti-beta 2m antibodies in SLE exert some inhibitory effect on antigen-induced T-cell proliferation, other distinct autoantibody systems to T-cell activation antigens appear to play the predominant role in this regard.     

Overestimation of myocardial infarct size by histologic measurement in a model of occlusion followed by reperfusion.

We studied 32 transverse left ventricular slices of myocardium from 16 pigs after 45 to 100 minutes of coronary artery occlusion followed by 180 minutes of reperfusion. Infarct area for each slice was determined as follows: (1) grossly, by triphenyl tetrazolium chloride staining of each slice, and (2) microscopically, by complete histologic sectioning of the triphenyl tetrazolium chloride-stained surface of each slice. Planimetry of necrotic and nonnecrotic areas was performed from tracings and photographs of triphenyl tetrazolium chloride-stained slices and from actual histologic sections. When triphenyl tetrazolium chloride and histologic measurements were compared, necrotic tissue area had decreased 11.4% +/- 15.0% (2.59 +/- 1.04 vs 2.09 +/- 0.86 cm2). Nonnecrotic tissue area decreased 20.6% +/- 24.0% (8.31 +/- 3.79 vs 5.16 +/- 2.73 cm2). In this model of ischemia followed by reperfusion, with fixation and processing, viable tissue shrank almost twice as much as necrotic tissue. This differential shrinkage introduces an error resulting in overestimation of infarct size by histologic quantitation.     

Intracoronary adiponectin at reperfusion reduces infarct size in a porcine myocardial infarction model

Reperfusion injury (RI) remains an important limitation of myocardial revascularization. The aim of the present study was to evaluate the influence of the intracoronary injection of adiponectin on RI and cardiomyocyte death in a porcine myocardial infarction model. Acute infarction in 14 Polish domestic pigs was induced by inflation of an over the wire balloon (OTW) catheter in the medial left anterior descending artery for 60?min. The study group consisted of 7 pigs in which intracoronary adiponectin (50?μg) was infused through the OTW catheter immediately before reperfusion. The control group (n=7) was administered placebo. Animals were sacrificed after two days of follow-up. The infarct area (IA) was stained with tetrazoline and the area at risk (AAR) with intracoronary administration of Evans Blue dye before euthanasia. Hearts in each group had similar AARs (46.2±9.9% vs. 48.4±6.2% of the whole myocardium, p=ns). The IA/AAR% and IA were smaller in the study group when compared to the control (24.7±4.0% vs. 45.3±22.5%, p=0.005; and 11.7±4.9% vs. 20.5±5.6%, p=0.01, respectively). These outcomes corresponded well with the peak troponin levels after 12?h (109.9±60.9 ng/ml vs. 185.5±39.4 ng/ml, p=0.017). After two days there was a significantly higher LVEF in the study group (51.4±8.5% vs. 33.9±8.6%, p=0.002). There was also a trend toward lower apoptosis enhancement in the viable myocardium in the study group (3.11±2.3 vs. 8.92±6.3; p=0.07). The administration of adiponectin into the infarct- related artery is safe and feasible. The treatment significantly reduced the infarct size.     

Enoxaparin, a low molecular weight heparin decreases infarct size and improves sensorimotor function in a rat model of focal cerebral ischemia

Possible neuroprotective effects of the low molecular weight heparin (LMWH) enoxaparin sodium (Lovenox) were evaluated in a rat model of focal ischemia. Male Sprague-Dawley rats were subjected to 90 min of occlusion of the right middle cerebral artery using the intraluminal suture method. Enoxaparin at doses of 0, 10 or 15 mg/kg was administered to groups of rats 1, 8, 24 and 32 h after artery occlusion. Motor impairment was evaluated by performance on the traverse beam and accelerating rotarod tests. Animals were sacrificed 48 h after occlusion and brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride for determination of infarct volume. Forty percent of the rats receiving 15 mg/kg enoxaparin died as a result of intracranial hemorrhage. Untreated rats exhibited large lesions involving the caudate putamen and much of the cortex. In enoxaparin - treated rats the damage was mainly confined to the caudate putamen. The sensorimotor behavior of the 10 mg/kg enoxaparin group was significantly better than that of untreated animals. Motor performance of the survivors in the 15 mg/kg group was poor due to hypoactivity and weakness resulting from excessive bleeding. These results suggest that LMWH may have a neuroprotective function.     

Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia

Background and purpose: We sought to determine the effect of vagus nerve stimulation (VNS) on infarct size after transient focal cerebral ischemia in rats. Methods: Ischemia was produced by transient filament occlusion of the right middle cerebral artery. Stimulating electrodes were implanted on the cervical part of the right vagus nerve. Electrical stimulation was initiated 30 min after the induction of ischemia, and delivered for 30 s at every 30 min for 3 h in experimental group 1 and at every 5 min for 1 h in experimental group 2. All the procedures were duplicated but no stimulus was delivered in the control group. Functional deficit was evaluated and animals were killed to determine the infarct size 24 h after ischemia. Results: Ischemic lesion volume was smaller in VNS-treated animals as compared with control animals; the relative percentage of contralateral hemispheric volume that underwent infarction was 16.2 ± 3.2% in the VNS and 33.0 ± 5.0% in the control arms in experimental group 1 (p < 0.05). The respective values for experimental group 2 were 19.8 ± 0.5% and 37.9 ± 2.6% (p < 0.05). VNS-treated animals were significantly more likely to have better functional scores at 24 h as compared with control animals. The functional score improved by 50% in experimental group 1 and 44% in experimental group 2 (p < 0.05 for both groups). Conclusion: VNS appears to offer protection against acute ischemic brain injury.     

Gene transfer of IkappaBalpha limits infarct size in a mouse model of myocardial ischemia-reperfusion injury

Nuclear factor-kappaB (NF-kappaB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IkappaBalpha prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IkappaBalpha gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV- IkappaBalpha) or the beta-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 10(11) copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 +/- 8%; IF/left ventricle = 44 +/- 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 +/- 0.4 micro g/gm tissue; infarct area = 2.9 +/- 0.6 micro g/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 +/- 0.4 relative amount of cardiac ICAM-1 mRNA). NF-kappaB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IkappaBalpha injection reduced infarct size (IF/area-at-risk = 19 +/- 3%; IF/left ventricle = 10 +/- 2%; p < 0.001), blocked NF-kappaB activation, diminished cardiac ICAM-1 expression (0.4 +/- 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 +/- 0.05 micro g/gm tissue; infarct area = 0.4 +/- 0.02 micro g/gm tissue; p < 0.001). Our data indicate that rAAV-IkappaBalpha may be useful for MI/R gene therapy.     

Effects of anti-beta 2-glycoprotein 1 antibodies and its association with pregnancy-related morbidity in antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by venous, arterial, or small-vessel thrombosis and/or pregnancy-related morbidity, associated with persistent positivity of antiphospholipid antibodies (aPL). Pregnancy-related morbidity in APS patients is characterized by unexplained fetal deaths, premature birth of morphologically normal newborns, and/or consecutive pregnancy losses before the 10^th week of gestation. Beta 2-glycoprotein 1 (ß2GP1) is the main antigen recognized by aPL and plays an essential role in the pathogenesis of APS. Antibodies against ß2GP1 (aß2GP1) are involved in damage-generating mechanisms in APS due to their interaction with trophoblasts, decidua, and endothelial cells. aß2GP1 might be used as a prognostic tool for obstetric risk stratification and ß2GP1 could be a target for molecular-targeted treatment to prevent pregnancy morbidity in APS. This review describes these aspects of aß2GP1, including effects on different cellular targets, its association with the severity of obstetric manifestations and the potential of ß2GP1-targeted therapies for APS.     

Anti-actin antibodies revealed by counter-immunoelectrophoresis. Relation to smooth muscle antibodies and bile canalicular antibodies.

In investigations by counter-immunoelectrophoresis, anti-actin antibodies were found in 59% of patients with chronic hepatitis and in 8% of patients with non-hepatic diseases and normal blood donors. Anti-actin antibodies were found more frequently in patients with hepatitis and IgG smooth muscle antibodies than in other groups of diseases and normal subjects with IgG smooth muscle antibodies. Anti-actin antibodies showed no correlation with bile canalicular antibodies.     

Role of anti-beta2 glycoprotein I antibodies in antiphospholipid syndrome: in vitro and in vivo studies

Antiphospholipid syndrome (APS) is characterized by the presence of recurrent venous/ arterial thrombosis and fetal losses associated with a family of auto-antibodies directed against phospholipid (PL)-binding proteins. Among them, beta2 glycoprotein I (beta2GPI) is the most important. As a plasma cationic protein, beta2GPI binds to anionic PLs involved in several fluid-phase coagulation steps, and more importantly, it can be expressed on the surface of different cell types. Anti-beta2GPI antibodies recognize the molecule expressed on endothelial cells, platelets, monocytes, and trophoblast cells. Once bound, the antibodies trigger in vitro cell signaling that modulates biological responses potentially responsible for pathogenic mechanisms. Experimental animal models have supported the in vivo pathogenic role of anti-beta2GPI antibodies in both thrombosis and fetal loss models.     

Expression of intercellular adhesion molecule-1 in rat heart with ischemia/reperfusion and limitation of infarct size by treatment with antibodies against cell adhesion molecules.

To elucidate the mechanism(s) of myocardial reperfusion injury, we investigated the roles of cell adhesion molecules on both leukocytes and vascular endothelial cells in the reperfused myocardia. We found that within 2 hours after reperfusion leukocytes began to infiltrate into the rat myocardia subjected to 30 minutes of ischemia and clarified, for the first time, that the expression of intercellular adhesion molecule-1 was enhanced on the capillary and venous endothelial cells from 8 to 96 hours after the start of reperfusion. Furthermore, pretreatment with individual monoclonal antibodies against cell adhesion molecules (CD11a, CD11bc, CD18, and intercellular adhesion molecule-1) reduced not only the infiltration of leukocytes but also the area of infarction in the reperfused hearts. These observations suggest that cell adhesion molecules play a critical role in the pathogenesis of myocardial reperfusion injury.     

Hyperbaric oxygenation pretreatment induces catalase and reduces infarct size in ischemic rat myocardium

Ischemia-reperfusion injury is a major complication occurring in heart stroke, cardiopulmonary bypass surgeries, and heart transplantation. Reactive oxygen species generated during the reperfusion phase overwhelm the scavenging capacities of antioxidant enzymes, and result in oxidative damage to the myocardium. We examined whether hyperbaric oxygenation (HBO) pretreatment induces antioxidant enzymes and protects the heart from subsequent ischemia-reperfusion injury. Rats were intermittently exposed to 100% O2 at 3 ATA (where ATA is absolute atmosphere) for 1 h daily and then sacrificed after 24 h of recovery in room air. Isolated hearts were subjected to 40 min of ischemia and 90 min of reperfusion. HBO pretreatment was found to condition the heart and enhance enzymatic activity and gene expression of catalase, thereby significantly reducing infarct size after reperfusion. A catalase inhibitor, 3-amino-1,2,4-triazole, completely abolished the infarct-limiting effect of HBO pretreatment, which suggests that HBO-induced tolerance against ischemia-reperfusion injury is due to catalase induction. Our results imply that HBO preconditioning may be developed as a new preventive measure for reperfusion injury in the heart.