Thromboembolic events and anti-tumor necrosis factor therapies.

Thromboembolic (TE) events have been observed in about 4.5% of patients treated with TNF antagonists. It has been suggested that anti-drug antibodies could be involved. However, another mechanism fits more with the available immunochemical data and could lead to practical measures to prevent TE events during anti-TNF therapies. Adverse effects are not related to the type of antagonist, but well to the combination of the inhibition of TNF and the predisposition of some patients to lupus-like reactions, including antiphospholipid syndrome. The overproduction of interferon-α, caused by the inhibition of TNF in these individuals would foster the development of lupus-like syndrome. Therefore, seeking conventional markers of systemic lupus erythematosus (e.g. anti-dsDNA, anti-phospholipid, anti-ß(2)-glycoprotein antibodies) before the administration of an anti-TNF could be a prudent measure.

Variability of the inhibition by total immunoglobulin of in vitro autoantibody-mediated erythrophagocytosis by mouse macrophages.

Several autoimmune diseases, mainly autoantibody-mediated, are attenuated by infusion of total IgG (IVIg). The efficacy varies widely from one patient to another. Using an experimental model of in vitro phagocytosis of autoantibody-coated erythrocytes by mouse macrophages, we analysed the possible causes for such a variability. Our results indicated that the efficacy of the phagocytosis inhibition depends upon different factors, such as the isotype and the extent of polymerization of the immunoglobulin used for the treatment as well as the genetic background of the mice and the state of macrophage activation that can be influenced by concomitant viral infection. The development of an in vitro assay for the phagocytic activity of macrophages might improve the selection of patients susceptible to benefit from IVIg treatment.

Exacerbation of autoantibody-mediated hemolytic anemia by viral infection.

Strong enhancement of the pathogenicity of an antierythrocyte monoclonal antibody was observed after infection of mice with lactate dehydrogenase-elevating virus. While injection of the antierythrocyte antibody alone induced only moderate anemia, concomitant infection with this virus, which is harmless in most normal mice, led to a dramatic drop in the hematocrit and to death of infected animals. In vitro and in vivo analyses showed a dramatic increase in the ability of macrophages from infected mice to phagocytose antibody-coated erythrocytes. These results indicate that viruses can trigger the onset of autoimmune disease by enhancing the pathogenicity of autoantibodies. They may explain how unrelated viruses could be implicated in the etiology of autoantibody-mediated autoimmune diseases.

Particle counting immunoassay for urinary cotinine. Comparison with chromatography, enzyme-linked immunoassay and fluorescence polarization immunoassay.

Urinary cotinine was measured according to its inhibitory activity on the agglutination of cotinine-coated latex particles by anti-cotinine antibodies, the agglutination being measured by optical counting of the remaining non-agglutinated particles (particle counting, PaC). The detection limit was 0.03 microgram/ml and the practical range extended from 0.03 to 3.9 micrograms/ml. The correlation results of 320 urine samples with those of high pressure liquid chromatography, enzyme-linked (Coti-Tracq EIA, Serex Inc., Maywood, NJ, USA), and fluorescence polarization immunoassay (TDX instrument, Abbott, Abbott Park, IL, USA) were r = 0.90, r = 0.69, r = 0.87, respectively, whereas the correlation coefficients between the assays other than particle counting ranged from 0.62 to 0.88. PaC does not require any separation step and can thus be easily automated.

Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p < 10(-4) and p = 0.006, respectively), by Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

The mode of action of treatment by IgG of haemolytic anaemia induced by an anti-erythrocyte monoclonal antibody.

In order to gain insight into the mechanisms by which the infusion of IgG can improve some autoimmune diseases, we induced haemolytic anaemia in mice by the injection of anti-erythrocyte MoAbs derived from NZB mice by S. Izui (Geneva). The IgG1 antibody 31-9D induces anaemia by erythrocyte sequestration in the spleen and liver, whereas the IgG2a antibody 34-3C triggers erythrophagocytosis (Shibata et al., Int Immunol 1990. 2:1133). Treatment of mice with pools of either human or mouse IgG clearly attenuated the anaemia induced by 34-3C, but not by 31-9D. Similar protection was obtained with human monoclonal IgGs from myeloma patients. Prior absorption by mouse erythrocytes did not affect the efficacy of the injected IgG. Treatment with Fc fragments also reduced the anaemia. In vitro experiments confirmed that 34-3C, but not 31-9D, triggered erythrocyte phagocytosis by murine macrophages. This process was completely inhibited by addition of polyclonal or myeloma IgG or of human Fc fragments. These results indicate that, in this model of autoimmune pathology, the protective effect of IgG is mediated by its interaction with the macrophage Fc receptors.

Influence of viral infection on anti-erythrocyte autoantibody response after immunization of mice with rat red blood cells.

Natural or deliberate activation of the immune system of pathogen-free mice markedly affected their response to an autoimmune-inducing stimulus. Specifically, mice immunized with rat red blood cells were found to make antibodies reactive with both rat and mouse erythrocytes. Animals housed for an extended period in a conventional environment developed an autoimmune response twice as fast as those kept in isolators. In an attempt to emulate this effect, mice kept in a sterile environment were infected with a potent polyclonal activator of B lymphocytes, lactate dehydrogenase-elevating virus, at the same time as they were inoculated with rat erythrocytes. Whereas uninfected animals developed a progressively increasing autoantibody titer, infected mice quickly attained high anti-erythrocyte autoantibody titers that remained rather constant. Contrary to circulating autoantibodies, bound anti-erythrocyte antibodies decreased with time. Virus infection enhanced all the IgG subclass responses, with the exception of IgG1, to both rat and mouse erythrocytes. None of the modifications of the autoimmune responses resulted in anemia.

Elimination of infectious antigens and increase of IgG catabolism as possible modes of action of IVIg.

Many mechanisms can explain the mode of action of IVIg in immune disorders. Macrophage blockade and interference in the idiotypic network are supported by some experimental data. Among the other mechanisms, two are considered in greater detail. Firstly, in some disorders, the patients could improve simply because the infused Ig contains antibodies directed against the infectious antigen causing the disease. Secondly, one can expect that IVIg increases the IgG catabolism and therefore the elimination of the autoantibodies. When the concentration of IgG in the plasma reaches 200% of the normal value, for example, the fractional catabolic rate increases up to 180% of its normal value. In other words, the half-life of IgG is decreased from 21 days to 12 days.

Latex agglutination assay of human immunoglobulin M antitoxoplasma antibodies which uses enzymatically treated antigen-coated particles.

An assay of immunoglobulin M (IgM) antitoxoplasma antibodies which is rapid (less than 30 min), homogeneous, and reliable (interassay coefficient of variation, less than 11%) is proposed. Its principle is based on the observation that a suspension of latex particles coated with toxoplasma antigens, after treatment with proteinase K, becomes less agglutinable by IgG antibodies but more agglutinable by IgM antibodies. The difference between the activities of the two classes of antibodies is increased by the addition of a monoclonal antibody directed against the Fc region of IgM. Agglutination is measured with a special instrument which optically counts the particles that remain free after the reaction. Turbidimetric reading, although less sensitive, is also suitable. No significant interferences either by IgG antitoxoplasma antibodies or by rheumatoid factor or antinuclear antibodies were observed. The sensitivity was similar to that of the immunosorbent agglutination assay.

Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

Human immune response to allergens of house dust mite, Dermatophagoides pteronyssinus. Isotypic analysis of antibodies in atopic and non-atopic subjects.

Specific antibodies isolated by immunoabsorption on four main insolubilized allergens from Dermatophagoides pteronyssinus (DPT) had the following isotypic distribution: in 16 atopic patients, 52% IgG, 40% IgM, 8% IgA, 0.1% IgE and, in 12 non-atopic individuals, 48% IgG, 46% IgM, 6% IgA, 0.03% IgE. The ratios between geometric means of antibody values in each class (atopic vs non-atopics) were 2.4 for IgG, 2.0 for IgM, 2.8 for IgA and 66.7 for IgE. The amount of anti-DPT antibodies in IgG subclasses did not follow the usual distribution of total IgG subclasses, i.e., IgG1 greater than IgG2 greater than IgG3 greater than IgG4. In atopics the order was IgG2 greater than IgG1 greater than IgG4 greater than IgG3 and in non-atopics, IgG4 greater than IgG1 = IgG2 greater than IgG3 although 6/12 of the latter had no detectable (less than 0.5 micrograms/ml plasma) IgG4.

Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine.

Immunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of Brucella abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibody-coated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma. CBA/H mice were infected with various doses [65-(65 x 10(6) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).

Immunoassay of hepatitis B surface antigen by particle counting after pepsin digestion.

Particle counting immunoassay is based on latex agglutination, the reaction being measured by instrument counting of the particles remaining unagglutinated. Most interference which generally affects latex agglutination can be avoided by pepsin digestion of the sample, provided the antigen (Ag) of interest resists pepsin, which is the case of the hepatitis B surface antigen (HBsAg). Pepsin treatment has the additional advantage of inactivating antibodies and so releasing the Ag from immune complexes. We have set up an assay of HBsAg, proceeding in a prototype of Impact Instrument (Acade Diagnostic Systems, Belgium) at a rate of 60 samples.h-1 and a total running time of 2 or 4 h. This assay was compared with Abbott radioimmunoassay (RIA) in 706 consecutive patients (A) and 31 selected sera for which values close to the cut-off had been obtained by RIA (B). In A, 38 sera were found positive and 668 negative by both methods. In B, RIA after neutralization classified the samples as positive (n = 14), negative (n = 14), or dubious (n = 3). Complete agreement between latex and RIA was achieved for nine positive, 12 negative, and two dubious samples. Of five RIA-positive samples, two were classified as latex-negative and three as dubious in the latex assay. One sample dubious in RIA was found latex-positive and two RIA-negative samples were found, respectively, latex-positive and dubious; when retested after pepsin digestion, the first of them became RIA-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoassay of rheumatoid factor by latex particle counting.

We assayed rheumatoid factor by instrumental latex particle counting. The calibration curve ranged from 12.5 to 500 int. units/mL. Maximal within- and between-assay CVs were 5 and 11%, respectively. Analytical recoveries ranged from 92.4 to 108%, and the relation between results and dilutions was linear in the range of 15 to 400 int. units/mL. Correlation with an enzyme-linked immunoassay (ELISA, Cordia kit) was r = 0.934 (n = 58), with turbidimetry r = 0.825 (n = 100), and with the Waaler-Rose test r = 0.834 (n = 73). Of 260 blood donors, 95% gave a value less than 10 kilo-int. units/L, which was taken as the upper normal limit. In a population of patients with rheumatoid arthritis (n = 47), 87.2% had rheumatoid factor greater than 10 kilo-int. units/L.

Lactoferrin uptake and iron processing into macrophages: a study in familial haemochromatosis.

Various properties of lactoferrin from neutrophils of normal individuals and patients with familial haemochromatosis were compared. No difference was found with respect to (1) the lactoferrin content of neutrophils, the molecular weight and isoelectric point of the protein, the dissociation of its complex with iron at acidic pH, its binding to isolated monocytes, and its uptake by the mouse reticulo-endothelial system. Macrophages from patients and controls were also found to be similar in their ability to bind and ingest lactoferrin and to process the iron provided by the protein. Therefore a defect in the interaction of lactoferrin with the reticulo-endothelial system, related either to the protein itself or to the cells, seems unlikely. A comparison of the lactoferrin- and the transferrin-mediated iron processing by monocytes is finally presented.

Particle counting immunoassay--an overview.

The principles and applications of the Particle Counting Immunoassay technique (PACIA) are summarized, and the limit of detection of the method is estimated. The various serum interferences are discussed and solutions to the problems proposed.

Pregnancy-specific beta 1-glycoprotein in serum in monoclonal gammopathies: relationship with serum beta 2-microglobulin, and cellular origin.

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay in serum from 86 healthy blood donors and 236 patients with various types of gammopathy. A concentration of 1 microgram/L was taken as the upper normal limit. Abnormally high values were found in one of 10 patients with monoclonal gammopathy of undetermined significance, in 65% of 152 patients with multiple myeloma, in 84% of 64 patients with Waldenström's macroglobulinemia, and in seven of 10 patients with monoclonal gammopathies associated with other myeloproliferative disorders. In a study of 90 myeloma patients, the SP1 value correlated (p less than 0.001) with the concentration of beta 2-microglobulin in serum, a value which had been corrected for possible renal dysfunction, but not with the concentration of the monoclonal component. SP1 was detected by direct immunofluorescence in myeloma cells of bone-marrow smears from six of 10 patients with myelomatosis. These six patients had serum SP1 values greater than 1 microgram/L, whereas the four patients with fluorescence-negative myeloma cells had SP1 values less than 1 microgram/L.

The human immune response against major allergens from house dust mite, Dermatophagoides pteronyssinus. II. Idiotypic cross-reactions of allergen-specific antibodies.

Antisera were prepared in rabbits against the idiotypic (Id) determinants of antiallergen antibodies. These antibodies were isolated from the plasma of 10 unrelated patients by immunoabsorption. Three major allergens isolated from the house dust mite, Dermatophagoides pteronyssinus (DPT), were used. The anti-Id antisera were rendered specific by successive absorption on insolubilized allergens, IgG and IgM from a pool of human sera and Ig from the donor after removal of anti-DPT antibodies by absorption. Anti-Id antibodies raised essentially against IgG antibodies were found to inhibit the agglutinating activity of both IgG and IgM anti-DPT antibodies toward allergen-coated latex. These anti-Id antibodies were also able to inhibit partly the binding of radiolabeled allergen to IgE anti-DPT antibodies. Matching the anti-Id antisera with the various individual anti-DPT antibodies revealed an Id cross-reactivity of about 71% for IgG, 61% for IgM and 62% for IgE. A second and predominant type of anti-Id antibodies recognized bystander idiotopes as the allergen did not inhibit Id-anti-Id reaction. These idiotopes were apparently more specific to individuals (private) as the anti-DPT antibodies of a given patient did not agglutinate latex particles coated with anti-Id antibodies prepared against Id of another patient.

Particle-counting immunoassay of a fetuin-like antigen in serum and cerebrospinal fluid.

A fetuin-like antigen was detected (smallest concentration detectable: 5 micrograms/L) by particle-counting immunoassay in 2% (13/641) of consecutive patients' sera but not in sera from 80 healthy blood donors, 40 neonates, or 40 pregnant women. The relation of the presence of detectable antigen to patients' diagnosis is not yet clear. However, in the group with cancer (154), it was found only in two of four patients with nephroblastoma and in three of five with tumors of tissue derived from the neurological crest: retinoblastoma (1/1), neuroblastoma (1/3), and medulloblastoma (1/1). Serum specimens from 422 patients with neurological disorders showed the antigen at a concentration greater than 5 micrograms/L in cases of neurosyphilis (5/11), peripheral neuropathy (12/38), Guillain-Barré syndrome (7/27), and multiple sclerosis (74/184). When we assayed 232 specimens of cerebrospinal fluid from the same neurological patients, we found the antigen in two cases of multiple sclerosis (6 and 15 micrograms/L) and in one case of Guillain-Barré syndrome (54 micrograms/L).