Lupus-like anticoagulant properties of murine monoclonal antibodies to β2-glycoprotein I

The lipid-binding inhibitor of coagulation, beta 2-glycoprotein I (beta 2GPI), has been shown to form the antigen to which some autoantibodies against anionic phospholipids (aPL) are directed. Six murine monoclonal antibodies (MAbs) of the IgG1 isotype were raised against human beta 2GPI and could be subdivided into three groups on the basis of mutual competition experiments. MAbs 9G1 and 8C3 (group A) markedly inhibited the binding of immunoglobulins from aPL-positive sera to beta 2GPI-coated wells. Using a lipid-based solid-phase radioimmunoassay, the MAbs interacted with both anionic phospholipids and phosphatidylethanolamine, but not phosphatidylcholine, in a beta 2GPI-dependent manner. A cross-reaction between beta 2GPI from several (including bovine) species was seen with one of the MAbs (9G1). All six MAbs induced dose-dependent prolongation of the DAPTT, DRVVT, KCT and TTI clotting times of human plasma, whereas 9G1 was the sole antibody to be inhibitory with plasma from bovine origin. Synergistic inhibitory effects were observed with MAbs used in pairs provided that they did not compete with each other for beta 2GPI binding. The anticoagulant activity of the MAbs was fully neutralized by the addition of freeze-thawed platelets. The MAbs described here resemble lupus anticoagulants in several respects which makes them valuable to study the involvement of beta 2GPI in the autoimmune thrombotic pathophysiology.     

β2-glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies

Some investigators have reported that a serum protein, beta 2-glycoprotein 1 (beta 2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to beta 2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to beta 2GP1 in the absence of cardiolipin. beta 2GP1 caused enhanced binding of aCL antibodies to cardiolipin, but this enhancement was not observed in inhibition experiments. Binding to cardiolipin occurred in the absence of beta 2GP1. Enhancement of cardiolipin binding activity by beta 2GP1 was observed for APS, but not for syphilis. We conclude that beta 2GP1 is not the antigen for aCL antibodies, nor is it likely that the antibody recognizes shared beta 2GP1-cardiolipin epitopes. Instead, this protein may make cardiolipin more available for aCL binding on solid surfaces by some yet undefined mechanism. This effect may not extend to aqueous suspensions.     

Renal Metabolism of β2-Microglobulin

beta 2-microglobulin (beta 2M) is a protein of 11,800 daltons which occurs in the plasma of normal individuals at concentrations of approximately 2 microgram/ml. It is presumed to be relatively freely filterable. More than 99% of the filtered beta 2M is taken up by an active reabsorptive mechanism and catabolized by the renal tubule. The data presented here demonstrate that renal extraction is only slightly diminished by complete ureteral obstruction. The renal extraction of beta 2M is greater than can be accounted for by filtration alone. These data indicate that some uptake of beta 2M occurs from the peritubular capillary circulation. The loading of animals with beta 2M is associated with a marked tubular proteinuria suggesting that this protein may play a part in inducing tubular injury.     

Significance of β2-Microglobulin in Liver Diseases

Serum levels of beta 2-microglobulin (beta 2-M) were found to be significantly elevated in acute viral hepatitis, chronic persistent or active hepatitis and liver cirrhosis. beta 2-M values were significantly lower in chronic persistent hepatitis than in the three other groups. Serum beta 2-M was normal in 75 asymptomatic carriers of HBsAg. Steroid therapy was followed by reduction of serum beta 2-M levels in 11 cases of chronic active hepatitis. Variations of beta 2-M were independent from that of transaminases, bilirubin and gamma-globulins.     

Pretreatment serum β2-microglobulin in multiple myeloma

Serum beta 2-microglobuline (S-beta 2m) was evaluated in 121 untreated patients with multiple myeloma. Values greater than 3 mg/l were found in 82% of the patients. Mean S-beta 2m values of the total group of patients correlated with clinical stage. However, there was no correlation if values were corrected for S-creatinine. Seventy-nine patients had normal (less than or equal to 106 mumol/l) and 52 patients abnormal S-creatinine. Patients with S-beta 2m values below 7 X 6 mg/l had an estimated median survival of 44 months compared to 12 months for patients with levels above 7 X 6 mg/l. If S-beta 2m values in patients with normal S-creatinine were combined with values corrected for S-creatinine from patients with elevated S-creatinine a beta 2m cut off level of 6 X 6 mg/l gave a median probable survival of 43 months compared to 14 months. We conclude that pretreatment S-beta 2 microglobulin is a useful marker for predicting survival in multiple myeloma. The problem of the relationship between S-beta 2m and S-creatinine is discussed.     

Haemoglobin Inkster (α285 aspartic acid → valineβ2) Coexisting with β-Thalassaemia in a Caucasian Family

S ummary . Haemoglobin Inkster, a new α-chain variant, was discovered in a family which also had the gene for β-thalassaemia. The amino acid abnormality was in αTp-9 which contains 29 amino-acid residues. Structural studies were facilitated by cleavage of the abnormal α-chains with cyanogen bromide followed by tryptic digestion. The substitution was shown to be valine for aspartic acid at position 85 in the α-chain. Affected individuals had no haematological abnormalities. Individuals with both β-thalassaemia and Hb Inkster had slightly lower percentages of Hb Inkster than those found in persons heterozygous for the Hb Inkster gene alone. 'Interaction' between thalassaemia and variant haemoglobin genes involving different haemoglobin loci has been reported in another family with β-thalassaemia and an α-chain haemoglobin mutant, as well as in the converse situation of coexisting β-thalassemia and a β-chain haemoglobin mutant. This decrease in the mutant haemoglobin percentage differs from the more common 'interaction' of thalassaemia and mutant haemoglobin genes involving the same haemoglobin locus, in which the mutant haemoglobin percentage is increased. The mechanism for the 'interaction' is unknown, but the presence of an unusually low percentage of a haemoglobin variant should warrant investigation for coexisting thalassaemia involving a different haemoglobin locus.     

β2-Microglobulin: Structure, Function and Significance

beta 2-Microglobulin is a low molecular weight protein with sequence homology to immunoglobulins. As a portion of the HLA complex this protein is an important cell-surface structure. Under normal conditions beta 2-microglobulin is synthesized and shed by many cells, particularly lymphocytes, and is detectable in the circulation of normal individuals. Because of its small size it is normally filtered readily at the glomerulus and is catabolized by proximal tubular cells of the kidney. Impaired renal function and hyperproduction of beta 2-microglobulin are both associated with increased serum levels. A function for beta 2-microglobulin as a modulator of lymphocyte surface and as a potential regulator of the immune system is proposed.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

Distinguishing β2-glycoprotein I dependent (systemic lupus erythematosus type) and independent (syphilis type) anticardiolipin antibody with Tween 20

Summary We investigated whether or not the use of Tween 20 could help to distinguish β₂-glycoprotein I (GPI) independent anticardiolipin antibody (aCL) (syphilis-type aCL) from GPI-dependent aCL (SLE-type aCL) in a GPI-dependent/ independent aCL ELISA. aCL was positive in all 16 SLB patients arid all 15 syphilis patients, who were positive for aCL in the standard ELISA. in the GPI-independent ELISA with Tween 20. GPI-dependent aCL was detected in 12/16 SLE patients by the GPI-dependent ELISA with Tween 20. aCL was not detected in any of the syphilis patients by GPI-dependent ELISAs. On the basis of these results, we recommend that Tween 20 should be used in ELISAs to distinguish GPI-dependent aCL from GPI-independent aCL.     

Long-term prognostic value of serum β2 microglobulin in myelomatosis

The prognostic value of serum beta 2 microglobulin (s beta 2m) measured at entry, at plateau and at 3-monthly follow-up times has been assessed for patients in the Medical Research Council's 4th and 5th Myelomatosis Trials. Analysis of 1014 patients confirmed the value of presentation s beta 2m of predicting survival in the short term but showed that predictive value was lost for subsequent survival in those patients who had survived for at least 2 years. However, measurements of s beta 2m taken during follow-up were predictive of subsequent survival. The predictive power of these follow-up measurements was very similar to that for the presenting measurement, and again they were only of value in predicting survival in the next 2 years.     

Demonstration of β2-Microglobulin in the Kidney by Direct Immunofluorescent Staining

Direct immunofluorescent staining with fluorescein isothiocyanate conjugated goat anti-human beta 2-microglobulin (beta 2M) was used to determine the localization of beta 2M in kidney tissue, obtained by biopsies or nephrectomies for various renal diseases. beta 2M was found in the renal tubular epithelial cell cytoplasm and occasionally in the tubular basement membranes in 48 out of 79 pathologic specimens tested, but not in 3 normals. beta 2M was seen in the tubular cells in all cases of primary tubulointerstitial disease and in all cases with secondary tubular damage associated with glomerular disease or systemic disease. This supports the concept that beta 2M is metabolized in proximal tubular epithelial cells, and its deposition indicates impairment of tubular handling.     

Haemoglobin α2β223Val → Ile produced in Escherichia coli facilitates Hb S polymerization

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.     

Resistance to activated protein C activity of an anti-/2 -glycoprotein I antibody in the presence of β-glycoprotein I

Some researchers claim that lupus anticoagulant-positive plasma may cause a false-positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis, We investigated whether anti-/3₂ -glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti-cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.     

Combined Cholinergic Antagonist and β2-Adrenoceptor Agonist Bronchodilator Therapy by Inhalation

Summary: The bronchodilator effects of 40 μg ipratropium bromide (I) and 400 μg fenoterol (F) by pressurised aerosol and both drugs in combination were compared with placebo (P) in a double-blind study in eight patients with chronic, partially reversible airways obstruction. The four treatments were (1) IP, (2) PF, (3) IF and (4) PP, with the second aerosol administered two hours after the first. Both drugs produced significant bronchodilatation for five hours, the response being greater and more rapid in onset with fenoterol. Both drugs in combination (IF) produced significant additive bronchodilatation from three to six hours after fenoterol. This additive effect may have been due to the improved lung function caused by ipratropium bromide and does not imply a synergistic effect. There were no side-effects reported. The results suggest that both ipratropium bromide and fenoterol are effective bronchodilating agents in patients with chronic asthma.     

Canine Ventricular Myocyte β2-Adrenoceptors Are Not Functionally Coupled to L-Type Calcium Current

INTRODUCTION: To establish the functional coupling of beta adrenoceptor (betaAR) subtypes beta1AR and beta2AR to L-type calcium current (I(CaL)), we investigated the nonselective agonist isoproterenol (ISO) and the relatively selective beta2AR agonists zinterol (ZIN) and salbutamol (SAL) on I(CaL) in isolated canine ventricular myocytes in the presence and absence of CGP 20712A (CGP) and atenolol (AT), selective beta1AR antagonists, and ICI 118,551 (ICI) a selective beta2AR antagonist. METHODS AND RESULTS: Peak I(CaL) was determined using "patch type" microelectrodes and whole cell voltage clamp. ISO (0.5 microM) increased I(CaL) maximally 3.5 +/- 0.67 fold. ZIN (10.0 microM) and SAL (10.0 microM) increased I(CaL) maximally 1.5 +/- 0.2 fold (n = 5) and 1.4 +/- 0.1 fold (n = 5), respectively. These effects were fully inhibited by CGP (0.3 microM) and AT (1.0 microM), which are inhibitors of beta1AR, but not by ICI (0.1 microM), which is a beta2AR inhibitor. ZIN at relatively lower concentrations (< or = 0.1 microM) did not increase I(CaL). CGP (0.3 microM) but not AT and ICI inhibited I(CaL) in the absence of betaAR agonists. CGP inhibition of I(CaL) was absent in the presence of forskolin (1.0 microM), which increases cAMP levels and I(CaL) by directly stimulating the adenylate cyclase. These data indicate that none of the antagonists affect I(CaL) through an action downstream of betaAR. CONCLUSION: Beta-adrenergic agonists increase I(CaL) via beta1AR but not beta2AR in canine ventricular myocytes.     

Acute Ethanol Intoxication Inhibits Neutrophil β2-Integrin Expression in Rats During Endotoxemia

The effects of acute ethanol intoxication on neutrophil [polymorphonuclear leukocyte (PMN)] adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol. Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. Control animals received an intraperitoneal injection of saline. Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 μg/rat in 2.5 ml of saline) or saline. Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 μg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion. Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats. Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs. Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats. Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication. Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs. G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis. The impairment of β₂-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection. G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.