Pertussis encephalopathy with high cerebrospinal fluid antibody titers to pertussis toxin and filamentous hemagglutinin.

A 7-year-old unimmunized girl with pertussis presented with respiratory failure and electroencephalographic evidence of an encephalopathy. The cerebrospinal fluid (CSF)/serum ratio of antibodies to pertussis toxin and filamentous hemagglutinin were 11- and ninefold higher than the CSF/serum ratio of total immunoglobulin G. The CSF/serum ratio of albumin was normal. These findings indicate production of antibodies in the central nervous system to Bordetella pertussis antigens and imply, therefore, that the pertussis encephalopathy in this girl was associated with the entry of pertussis antigens into the central nervous system.

A simple two-step procedure for the purification of plasma C1q from different animal species.

Plasma C1q from human, rat, rabbit, dog and sheep were isolated by a 2-step affinity chromatography procedure. In the first step the method exploits the affinity of C1q for heparin and in the second the interaction between C1q and IgG. The precipitation of C1q by the SO4(2-) groups in agarose gels was used as a means to rapidly monitor the elution of C1q. This interaction was found to be non-species specific and therefore obviates the need for immunochemical and/or haemolytic assays. The isolation procedure is rapid, simple and is not confined to one species. Pure functionally active C1q was obtained from all species in yields of approximately 85-95%.

Elution of anticardiolipin antibodies and their cofactor beta 2-glycoprotein 1 from the placentae of patients with a poor obstetric history.

Anticardiolipin antibodies (aCL) were eluted from the placentae of four women with elevated serum levels of aCL, demonstrating that these antibodies are bound to affected placentae. Anticardiolipin antibodies bound to affected placentae were only of the IgG isotype and the level of aCL in placental eluates did not reflect serum levels. Anticardiolipin antibodies were not isolated from placental eluates of control normal pregnancies. beta 2-Glycoprotein 1, the anticardiolipin antibody cofactor, was present in the placental eluates from both control and antiphospholipid antibody (aPL) affected pregnancies and was localised in the syncytiotrophoblast by immunohistochemical analysis. Antinuclear antibodies were present in the placental eluates of 3 of the 4 patients with antiphospholipid antibody syndrome and were absent from the placental eluates of control pregnancies. The authors propose that anticardiolipin antibody binds directly to placental tissue, disrupting uteroplacental blood flow and/or transport through the villi.

The effect of human anticardiolipin antibodies on murine pregnancy.

Anticardiolipin antibodies (aCL) were affinity purified or isolated in the IgG fraction of serum from 6 patients with antiphospholipid antibody syndrome. Anticardiolipin antibodies from one patient consistently compromised murine pregnancy. However in 92% (45 of 49) of cases injection of human anticardiolipin antibodies had no adverse effect on murine pregnancy, regardless of whether affinity purified aCL or IgG fractions were used. It is concluded that in most cases human anticardiolipin antibodies alone do not induce murine fetal loss.

Inhibition of heparin/antithrombin III cofactor activity by anticardiolipin antibodies: a mechanism for thrombosis.

Anticardiolipin antibodies (aCL) are autoantibodies which react with negatively charged phospholipids and are associated with thrombotic disease and recurrent fetal loss. We have found that 11% of aCL are cross-reactive with the glycosaminoglycans heparin and heparan sulphate. One of these antibodies was studied in detail and was found to inhibit the heparin dependent activation of antithrombin III by up to 80%. The inhibition of heparin dependent antithrombin III activation represents a new mechanism by which anticardiolipin antibodies may induce thrombosis or fetal loss in some patients with these antibodies.

Cofactor dependent and cofactor independent anticardiolipin antibodies.

Two distinct types of anticardiolipin antibodies are described, one of which requires the presence of a serum factor (cofactor) to bind cardiolipin in ELISA and liposome affinity systems. The second type does not require this cofactor. The requirement of a cofactor for the binding of some but not all anticardiolipin antibodies provides an explanation for the confounding variability of the results of assays for these antibodies. It also explains why blocking agents such as BSA and gelatin do not produce consistent results in this assay.

Practical requirements for immunochemical analysis of antithrombin III in agarose gels.

This report emphasizes the importance charged groups affixed to the agarose gel matrix have on immunoprecipitation of antithrombin III (AT) and of its protease complexes. Plasma and serum samples were analyzed for antithrombin III by electroimmunoassay and single radial immunodiffusion in different types of agarose. The results were compared with the amount of functionally active AT contained in the samples. A high correlation between the results was obtained provided the following criteria were fulfilled: (1) agarose with a sulphate content of 0.2% or more was used; (2) the antiserum should contain antibodies directed to AT plus antigenic determinant(s) that emerge(s) during the AT-protease interaction; (3) a gel buffer of about 75 mmol/l was used. Heparin added to the test system competed with sulphate groups affixed to the agarose gel matrix and inhibited immunoprecipitation of AT.

Enzyme-linked immunosorbent assay for ileal human goblet cell mucin.

A sandwich ELISA for measuring ileal human goblet cell mucin has been developed with a linear response range 0.2 to 1.5 ng mucin protein. It can be used to quantitate the mucin present in dilute and unpurified samples without interference from other glycoproteins and proteins. Reduction of the mucin decreased the reactivity by only 14% indicating that the assay reacts almost as well with mucin glycopeptides as with native mucin. The assay has the advantages over previously described immunoassays for mucin of giving a result in 6 h, detecting slightly lower concentrations of mucin, and is more sensitive, quantitative and specific than the traditional protein or periodic acid-Schiff assays used for glycoproteins.

Comparison of tests for the lupus anticoagulant and antiphospholipid antibodies in systemic lupus erythematosus.

A variety of laboratory assays are used to screen for the presence of the lupus anticoagulant. Six commonly used coagulation tests, and the ELISA assay for antiphospholipid antibody using three different substrate phospholipids, have been evaluated in 110 patients with systemic lupus erythematosus or lupus-like disease. One or more coagulation assays was abnormal in 41% (45/110) of the patients. No individual test detected more than 78% of these abnormalities, indicating that a single phospholipid based coagulation test cannot be used to screen for a possible lupus anticoagulant. A combination of Actin FSL, DTTA and DRVVT detected all the abnormalities. The most sensitive two-test combination was Actin FSL and DRVVT. Approximately half (56%) of the patients with a positive clotting test had an abnormal antiphospholipid antibody assay. A similar proportion (58%) of the aPL positive patients had a prolonged coagulation test. The marked discordance between the coagulation assays and a positive antiphospholipid antibody test further complicates the laboratory definition of this abnormality, at least in patients with systemic lupus erythematosus.

Location and secretion of gastric intrinsic factor in the sheep.

The site of production and secretion of intrinsic factor (IF) in the sheep has been studied using a human auto-antibody directed against IF. Immunofluorescent studies indicated the abomasal parietal cell was the source of IF in the sheep. Concentrations of IF in pure gastric secretion of sheep were relatively stable at 3 to 4 iu/ml and it was estimated that the total abomasal output of IF was 10,000 to 23,500 iu per 24 hours.

The measurement of complement as an aid in diagnosis: 1. Nephritis and collagen disease.

A selection of patients with nephritis and collagen diseases were studied for complement abnormalities. This communication briefly reviews the current comcepts of complement physiology, details standard assay used in this laboratory and describes clinical examples where these tests have provided significant clinical information. Quantitative measurements of individual components, the presence of circulating immune complexes, and the demonstration of in vivo C3 cleavage products were assessed simultaneously. The importance of measuring cleavage products and immune complexes is stressed. Clinical examples are given where misleading or erroneous interpretation of results might have occurred if these tests had not been performed.

Complement in acute leukaemia: its role in infection.

This study determines the frequency of complement system dysfunction in adult patients with acute myeloid leukaemia and its prognostic significance. Twenty four patients with acute leukaemia had complement studies performed at time of presentation and during the haematological trough occurring between 7 and 14 days after the administration of chemotherapy. At presentation 32% of patients had abnormal complement function. Only 54% of patients maintained normal complement activity throughout the period of remission induction. The aetiology of the disturbance is unclear and no single causative factor was determined. Patients with abnormal complement function had significantly greater morbidity from infection during the period of remission induction as measured by the number of febrile (greater than 38 degrees C) days (p less than 0.05), and were more likely to have pathogens isolated by blood culture during septic episodes (relative risk 4.0, p less than 0.01). These findings show a significant increase in morbidity associated with depressed complement function and emphasise the important role the complement system plays in maintenance of host defences.

Anticardiolipin antibodies: their presence as a marker for lupus anticoagulant in pregnancy.

A solid phase ELISA was developed to investigate the association between anticardiolipin antibodies and lupus anticoagulant in pregnancy. Twenty-seven pregnant women with a history of recurrent fetal losses or systemic lupus erythematosus (SLE) were tested for the presence of lupus anticoagulant, anticardiolipin antibodies, antinuclear antibodies (ANA) and anti-single-stranded DNA antibodies. Nineteen women with a total of 49 previous unsuccessful pregnancies were found to have lupus anticoagulant and anticardiolipin antibodies. Three women who had suffered four fetal deaths from six pregnancies had anticardiolipin antibodies without lupus anticoagulant. Cardiolipin antibodies were not detected in the remaining five patients. This assay for measuring anticardiolipin antibodies appears to provide a simple and inexpensive method of identifying women at risk of fetal death from the adverse effects of lupus anticoagulant.

The basidiomycete ganoderma and asthma: collection, quantitation and immunogenicity of the spores.

Ganoderma fungal spores are a major component of the Auckland air-spora. Previous studies of ganoderma involvement in allergic asthma and rhinitis were extended by locating the sporophores (fruiting bodies) in the Auckland area and systematically collecting the ejected spores. Maximum production by one sporophore was 5 gram dry weight of spores in one week, equivalent to 11 billion spores. We have estimated that between 400 and 1200 sporophores would account for previously reported levels of ganoderma spores collected from the air by Burkhard spore traps. Both whole spores and extracts of spores were strongly immunogenic in rabbits. Of the 115 asthma patients who were skin prick tested with a variety of fungal extracts, 32 (28%) were positive to one or more fungi. Of these, 18 (16%) reacted positively to ganoderma extracts. A theory proposing how ganoderma might contribute to allergic hyperreactivity in susceptible patients is developed.

Immunochemical analysis of active and inactive antithrombin III.

Antithrombin III (AT) levels from normal and AT deficiency persons were measured by electroimmunoassay (EIA) and the results compared with a chromogenic assay (S2238). Discrepant results were obtained when plasma and serum were compared using one antiserum, and therefore did not always relate to functional activity. Another antiserum, however, when used was capable of differentiating active AT from inactive AT complexed with its proteases and demonstrated close correlation with all samples tested (r=0.97). The specificity of the antisera and consequent anomalous results were elucidated when purified human thrombin was added to plasma samples and subsequently reanalysed. Quantitative differences observed when serum samples were compared by single radial immunodiffusion and electroimmunoassay with one antiserum, illustrates the fundamental principle differences between the two methods. These results give some insight as to why previous anomalies with AT immunoassays have occurred. They also indicate that plasma and not serum should be used as clinical test material. AT antisera should be capable of recognizing and distinguishing free AT from AT/protease complexes if the results obtained by electroimmunoassay are to correlate with functional activity.

The interaction of heparin with plasma proteins. Demonstration of different binding sites for antithrombin III complexes and antithrombin III.

Affinity chromatography of plasma and serum with the use of heparin conjugated Sepharose has confirmed the existence of two types of protein binding sites. A minor heparin fraction binds free AT III selectively and firmly but not its protease complexes. The complexes bind less firmly to another, much larger heparin fraction together with a select group of the plasma proteins at physiological pH and ionic strength. These included complement proteins (C1q, C2, factor B, properdin, and beta 1H), protease inhibitors (inter-alpha-trypsin inhibitor and C3b inactivator) and cell surface proteins (protein HC and fibronectin) as well as beta-lipoproteins. The results demonstrate that affinity chromatography with heparin-Sepharose is extremely useful as an early preparative step in the isolation of these minor plasma components. The results also indicate that the said proteins can be linked to cell surfaces carrying heparinoids in the intercellular space.