Immunity to schistosomiasis mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae: I. Humoral responses against skin-stage schistosomula.

The anti-schistosomular humoral responses of guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosoma mansoni have been investigated in vitro. The sera of vaccinated animals contain schistosomulicidal complement-fixing antibodies which peak in titre at week 5 after vaccination and predominantly consist of IgG2 and IgM antibodies. The ability of the serum to arm macrophages from normal animals to bind to schistosomula, also peaks in titre at week 5 and is associated with IgG2 antibodies. Basophils from normal animals can be sensitized in vitro by vaccine serum to degranulate in the presence of schistosomular antigens. This anaphylactic antibody activity is associated with IgG1 but not IgE antibodies, and peaks in titre at week 10. Three antigens (14 kD, 20 kD and 43 kD) are specifically and transiently detected by vaccine serum on Western blots of schistosomular proteins; these antigens are first discernible at week 4, but were virtually undetectable at week 12.     

Effect of Normal Human Serum on the Binding of Specific Antibodies and Platelet-unrelated Immune Complexes to Human Platelets

Radiolabelled staphylococcal protein A was used to quantitate the binding of IgG on stored human platelets from human sera containing specific antibodies reactive with platelets and rabbit serum containing immune complexes (IC). Normal human serum (NHS) inhibited the binding of IC onto platelets and to various extents also the binding of specific antibodies. The attachment of inhibitors to platelets seemed to be reversible. The considerable difference in the inhibitory capacities of IgG-deficient sera and monomeric IgG indicates that IgG is the major inhibitory component of NHS. The binding of IgG from NHS onto platelets evidently hampers the detection of weak platelet antibodies even with the most sensitive tests. Purified Clq, known to modify the reactions of IC with fresh platelets did not alter the binding of IC onto stored platelets. A monoclonal, antiglobulin-active rheumatoid factor of IgM class displayed only moderate inhibition. Therefore, the application of RF or Clq for the differentiation of the binding induced by IC or antibodies is not useful in this assay system. The heterogeneity of immunologic receptors of platelets provides an explanation of the inhibitory inefficiency of Clq.     

Antibodies selectively reactive to autologous and host-type platelets are obtained following interspecies immunizations in marmosets.

Interspecies immunization of marmosets with platelets leads to an acute thrombocytopenia which appears to be immunologically mediated. The thrombocytopenia is preceded by the formation of antibodies to the donor platelet antigen and deposition of IgG on the host's platelets. Examination of sera obtained from these animals during the course of the disease has revealed the presence of antibodies towards autologous and host-type platelets. Autologous antibody was found when thrombocytopenia was severe; upon platelet recovery, this antibody was absent or reduced in titre. This in vivo absorption of host-specific (autologous) antibody led to detection in the serum of antibodies reactive to other members of the host species. The presence of such host-type antibodies appeared to be related to the intensity of antigenic stimulation, the amount of antibody formed and the degree of thrombocytopenia. Deposition of IgG on the host's platelets coincided with the appearance of the anti-host antibodies; whether the IgG detected on the host's platelets is exclusively cross-reactive antibody or includes also an immune complex is not yet known. Finally, the data have identified potential immunogenetic differences among members of two marmoset species with respect to platelet antigens.     

Comparison of structural polypeptides, detected by immunoblotting technique, in the sera of spotted fever group rickettsia positive cases--symptomatic versus asymptomatic.

In an attempt to characterize the nature of symptomatic versus asymptomatic spotted fever group rickettsia (SFGR) infection, the immune response to R. conorii (boutonneuse fever) structural polypeptides was studied by Western-blot immunoassay. Sera from immunoperoxidase assay (IPA), SFGR seropositive (titre greater than or equal to 80) individuals, symptomatic and asymptomatic and from SFGR seronegative (IPA titre less than 80) individuals living in a kibbutz community in the desert region of Southern Israel were examined by immunoblot. This community suffered from a very high morbidity rate due to SFGR (21-fold higher than the national reported average). The entire community (n-326) has been followed-up since 1985, with serial serum samples being examined for specific IgG antibodies by IPA. The intensity of the immunoblot reaction correlated with specific IgG antibody titres as determined by IPA. This correlation was also observed between the decrease in the IgG titre and the strength of the antibody-antigen reaction by immunoblot over time for a given individual. IPA seropositive sera from asymptomatic as well as symptomatic spotted fever cases reacted to 8 individual polypeptides. In both cases antibodies to 22 kD, 24 kD, 26 kD, 28 kD, 30 kD, 32 kD, 34 kD, and 37 kD were found. In the IPA seronegative sera, antibodies to polypeptides in the range of 24 kD to 32 kD were not detected. The lack of detectable differences by immunoblotting between SFGR symptomatic vs, asymptomatic cases might be explained by other aspects of the immune response of each infected individual, and/or it is possible that virulent and non-virulent antigenically closely related SFGR strains infected symptomatic vs. asymptomatic individuals.     

Factor V: a platelet cytoskeletal associated protein

Platelet cytoskeletons prepared from thrombin activated platelets contain specifically associated Factor Va. We postulate that Factor Va associates with the cytoskeleton through receptors present on the platelet surface. The following evidence supports this hypothesis. (a) Prior secretion of Factor Va is necessary for the association of Factor Va with the cytoskeleton. (b) Reagents that inactivate Factor Va on the surface of the platelet such as EDTA and proteolytic enzymes also inactivate the Factor Va on the cytoskeleton. (c) The platelet Factor Xa binding sites (i.e. Factor Va) are quantitatively retained on the platelet cytoskeleton. (d) Platelet cytoskeletons prepared from platelets that are thought to be deficient in Factor Va binding sites contain 25 percent of Factor Va activity of normal platelet cytoskeletons and platelet cytoskeletons prepared from platelets deficient in Factor Va contain no Factor Va activity but regain control levels of Factor Va only when Factor Va is added to the platelets prior to the preparation of cytoskeletons.     

Platelet antibodies in idiopathic thrombocytopenic purpura.

An immunofluorescence (IF) technique for the detection of antibodies was applied to idiopathic thrombocytopenic purpura (ITP). Serum platelet antibodies were found in thirteen out of twenty-two patients (59 percent) with active disease, but in only four out of fifteen patients (27 percent) who had attained remission. Direct tests for platelet-associated IgG were positive in 36 and 44 percent of these patients respectively. In two cases IgM was observed on the patients' platelet membranes. C3 was not detedted on patients' platelets. Platelet-associated IgG was also found in several other disorders and its occurrence is not therefore diagnostic of ITP. In addition, serum platelet antibodies do not indicate specifically ITP as they may also be due to previous isoimmunization. Antibodies in the sera of patients with ITP generally did not fix Clq and in most cases bound to platelets only in the presence of EDTA. In contrast, isoantibodies often fixed Clq and they had equal affinity for platelets suspended in ACD or EDTA plasma. This was confirmed by quantitative data on IgG binding by platelets obtained by measuring 125-I-labelled protein A uptake. The simplicity of the IF technique permits its routine application and the technique may give useful information with respect to the nature of the antibodies. It must, however, be considered of limited value in the diagnosis of ITP.     

Anti-fibrillarin autoantibodies in mercury-treated mice

Using indirect immunofluorescence (IF) with HEp-2 cells as a substrate serially bled SJL mice were found to gradually develop a high titre of anti-nucleolar antibodies (ANuA) after 3-5 weeks of s.c. injections of 1.6 mg HgCl2/kg body weight every third day. The ANuA showed a clumpy nucleolar pattern of localization and were composed of all IgG subclasses, but contained, in comparison with the antinuclear antibodies (ANA) in MRL-lpr/lpr mice, significantly lower titres of IgG2a and only traces of IgG3. Immunoblotting analysis using purified mouse liver nucleoli revealed that the sera with ANuA identified the same 34-kD nucleolar protein which was targeted by a human scleroderma serum containing autoantibodies monospecific for fibrillarin. In addition, a fraction of the mercury-treated SJL mice developed serum antibodies reacting with 10-15 and 60-70 kD nucleolar proteins in immunoblotting. The presence of serum autoantibodies reacting with the 10-15 kD proteins correlated with significantly increased titres of anti-histone antibodies of the IgG class in ELISA. Some mercury-treated SJL mice also developed a significantly increased titre of anti-histone antibodies of the IgM class. B10.S mice treated with mercuric chloride consistently developed ANuA, which also targeted a 34-kD nucleolar protein. Since anti-fibrillarin antibodies are specific markers of scleroderma, the present animal model may be valuable for studies of the immunological aberrations which are likely to induce this autoimmune response.     

Microplate enzyme immuno-assay for detection of platelet antibodies

A simplified and sensitive enzyme immuno-assay employing microtiter plates as the solid phase carrier for detection of circulating platelet antibodies and bound antiplatelet IgG has been described. The assay was done using fresh as well as frozen platelets and commercially available peroxidase labeled anti-human IgG. The sensitivity of the enzyme immuno-assay was found similar or superior to that of the platelet suspension immunofluorescence test and superior to the lymphocyte cytotoxicity test and the platelet complement fixation test. The use of platelets frozen in the wells of the microtiter plates (stored for up to 17 months at -20 degrees C without loss of antigenicity) facilitates the performance of the test. In addition the small volumes of sera and platelets needed in using microtiter plates makes the assay particularly suitable in testing platelets from thrombocytopenic patients. In one of 31 sera from normal Zwa-negative donors a weak anti-Zwa (P1A1) was found only detectable by enzyme immuno-assay. Alloantibodies were found in 14 of 23 patients suffering from non-haemolytic transfusion reactions. All of three patients with post-transfusion purpura had anti-Zwa antibodies in serum. Anti-Zwa antibodies were demonstrated in the sera from all of 9 Zwa-negative mothers, who had given birth to children with alloimmune neonatal thrombocytopenia. In 8 of 12 Zwa-positive mothers alloantibodies of other specificities were found in the serum. In one case the antibody was a known anti-Baka. Six of 7 patients with autoimmune thrombocytopenic purpura had values of platelet-bound IgG exceeding the normal range and circulating platelet antibodies were seen in 4 patients.     

Use of the immunoblotting method in serodiagnosis of M. kansasii mycobacteriosis]

The indirect enzyme test ELISA with soluble complex antigen of M. kansasii, used for assessment of the titre of IgG serum antibodies in a group of patients suffering from mycobacteriosis M. kansasii and in a control group of patients suffering from tuberculosis did not reveal statistically significant differences between serological responses of these two groups. On the other hand, the immunoblot analysis revealed in sera selected at random from these two groups differences at the level of subprotein units against the complex antigen of M. kansasii. In a group of 15 sera of patients with M. kansasii the immunodominant area was 42-45 kD protein subunits, in the control group of 10 sera with M. tuberculosis the area was 35-39 kD. The western blot technique is more perspective for improvement of the serum diagnosis of M. kansasii, in particular where a specific antigen is not available.     

Serum-induced suppression of interferon (IFN) activity. Lack of evidence for the presence of specific autoantibodies to IFN-alpha in normal human sera.

IgG antibodies binding to different IFN species have been described in sera of healthy and diseased individuals. Human serum immunoglobulins have also been shown to interfere with IFN bioactivity. To characterize these antibodies, human recombinant IFN-alpha 2A (rIFN-alpha) was radioiodinated, and ligand binding studies were performed in human sera as well as on the human cell line A-549 in the presence of human serum. 125I-rIFN-alpha bound to serum factors of healthy individuals. However, less than 3% of the binding was to IgG and the binding was non-saturable and therefore most likely non-specific. 125I-rIFN-alpha bound to receptors on A-549 cells, and the ligand-receptor complexes appeared to internalize. However, both cell binding and internalization of 125I-rIFN-alpha were independent of the presence of human serum. We conclude that normal human sera do not contain detectable autoantibodies to rIFN-alpha.     

Individual variation of anti-Sm autoantibodies in the MRL/MP-lpr/lpr mouse: age-related increase in diversity.

The prevalence and diversity of anti-Sm antibodies (which are a marker of human lupus) were investigated in MRL/MP-lpr/lpr mice. The low prevalence of anti-Sm reported by other workers was confirmed. Anti-Sm antibodies bound to either the 28 kD or both 28 and 16 kD polypeptides on immunoblots. Antibodies to the 16 kD polypeptide, detected on spectrotyping blots, were not detected on immunoblots probed with sera from three mice, suggesting that these antibodies were binding only to an additional conformational epitope. Isoelectric focusing of the sera showed that the anti-Sm antibodies were restricted in clonal diversity and in some cases resembled the pattern of a monoclonal antibody. A longitudinal increase in antibody titre was correlated in some mice with an increase in the diversity of clones expressing anti-Sm antibody. Anti-nRNP antibodies were also detected in the mouse serum but had a lower prevalence than anti-Sm antibodies.     

Western blot analysis of water-soluble wheat flour (Triticum vulgaris) allergens.

Water-soluble wheat flour allergens were analyzed using the western blot technique. The sera of 130 bakers (42 with wheat flour allergy, 88 without allergy) were analyzed for IgE, IgG, and IgG4 binding towards wheat flour allergens. Three major allergens with molecular weights of 47, 17, and 15 kD were identified (Tri v Bd 47, Tri v Bd 17, and Tri v Bd 15; IUIS allergen nomenclature) by their ability to bind IgE from approximately 50% of the allergic sera. The specificity of the IgG was mostly directed against three high molecular weight components (134, 122, and 98 kD). In contrast, IgE and IgG4 was found frequently against the major allergens. 40% of healthy donors versus 12% of the asthmatic bakers had no detectable IgG4 against wheat flour extract. The biological activity of the water-soluble wheat flour components has been shown by their ability to induce histamine release from basophil leukocytes obtained from patients with bakers' asthma. The release of histamine from basophil leukocytes significantly correlated with the presence of IgE antibodies against wheat flour components of 47, 17 and 15 kD. A regulatory role of wheat flour specific IgG4 in allergic diseases could not be evaluated by western blot analysis.     

Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus

The isotype profile, particularly emphasizing IgG subclass distribution, of dsDNA antibodies in patients with systemic lupus erythematosus was evaluated using an especially adapted ELISA technique. Anti-dsDNA antibodies were quantified with class-specific antisera and subclass-specific monoclonal antibodies. IgG subclass specificity was proven with 20 myeloma proteins differing in light chains and allotypes. The standardization with myeloma proteins proved to be useful and reliable. Results from more than 100 anti-dsDNA positive sera from SLE patients showed specific antibodies within the three subclasses (IgG1: 52-100%, IgG2: 0-39%, IgG3: 0-48%). IgG4 was not detected in significant amounts. No correlation to the subclass distribution of total IgG was found. Each patients' serum displayed an individual isotype pattern that remained constant in longitudinal studies, independent of anti-dsDNA titre fluctuations. These results suggest a stable population of autoreactive clones in the progress of the disease.     

Autoimmunity to a 28-30 kD cell membrane DNA binding protein: occurrence in selected sera from patients with SLE and mixed connective tissue disease (MCTD).

Previous experiments have established the presence of a 30-kD DNA binding protein on the surface of human leukocytes. Herein we report that selected sera from patients with systemic lupus erythematosus (SLE) and MCTD are reactive with a 28-30 kD protein on immunoblots of peripheral blood mononuclear cells (PBMC) cell membrane preparations; the reactivity is abolished by prior incubation of the blot with DNA. Antibodies eluted from the 28-30 kD strip inhibited the binding of 3H. DNA to human PBMC. An immunomatrix of 28-30 kD reactive immunoglobulins was able to extract a 29-kD DNA binding protein from a PBMC cell membrane preparation. Flow cytometry experiments confirmed the cell surface IgG reactivity of sera with T lymphocytes. Additional experiments indicated that cell surface IgG binding was not due to antibodies binding to cell surface DNA, DNA anti-DNA immune complexes reacting with a DNA binding protein, anti-histone antibodies or anti-Sm antibodies. It is hypothesized that this autoimmune response could be one component of an idiotypic network involving anti-DNA antibodies.     

A solid phase assay with radioactively-labelled antibody for the detection of Brucella abortus.

An assay for the detection of Brucella abortus is described. IgG from rabbit antisera against live brucellae or brucella extracts was chemically linked to cellulose to form a solid phase reagent capable of binding brucella antigens present in buffer solutions or serum. After washing away any unbound material the presence of bound antigen was revealed by incubation with radioactively-labelled anti-brucella antibodies. The assay was capable of detecting less than 100 pg brucella antigen in a 20 microliter sample. Experiments in which IgG of non-related specificity was used in place of anti-brucella IgG showed that the test was specific. Normal human serum had only a slight inhibitory effect but anti-brucella antibodies were strongly inhibitory if present in the test sample. The extent of this effect and its relationship to antibody titre was investigated in 12 sera from brucellosis patients.     

Activation of human platelets through gp140, the C3d/EBV receptor (CR2)

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab')2 fragments.     

Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis.

The binding specificities were investigated of anti-phospholipid antibodies derived from sera from 55 patients with SLE and related diseases, and from 33 patients with syphilis. Antibodies from both these groups of patients bind strongly to cardiolipin in solid-phase immunoassays, but only antiphospholipid antibodies from patients with autoimmune diseases are associated with thrombotic complications and recurrent spontaneous abortions. IgG anti-phospholipid antibodies from both groups of patients cross-reacted with a range of negatively charged phospholipids, but binding to neutral phospholipids was largely restricted to sera from patients with syphilis. A monoclonal IgM lambda anti-cardiolipin antibody, derived from a patient with autoimmunity, was used to inhibit binding of anti-phospholipid antibodies to cardiolipin and to phosphatidic acid. This antibody inhibited the binding of autoimmune sera to cardiolipin more strongly than sera from syphilis patients, but the converse pattern of inhibition of binding to phosphatidic acid was observed. The VDRL titre correlated with anti-phospholipid antibody activity in sera from syphilis patients, but not from those with autoimmunity. Lupus anti-coagulant activity correlated weakly with IgG antibody levels to each of the negatively charged phospholipids among the patients with autoimmunity. Lupus anticoagulant activity did not correlate uniquely with any anti-phospholipid antibody specificity. These results provide further documentation of the great heterogeneity of anti-phospholipid antibodies associated with autoimmune disease and syphilis.     

Specificity of autoantibodies to histone H1 in SLE: relationship to DNA-binding domains.

Autoantibodies in the sera of patients with systemic lupus erythematosus were examined with respect to their specificity for proteolytic fragments of histone H1 that retain, or do not retain, DNA-binding domains. 16 of 31 sera contained IgG and IgM antibodies to histone H1. IgM antibodies to H1 in 8 sera (50%) were directed at 18 kD and 20 kD alpha-chymotrypic H1 fragments that bore binding sites for DNA, as identified by staining immunoblots containing the fragments with ssDNA plus 6/0, a mouse monoclonal antibody against ssDNA, IgM with this type of histone H1 specificity did not react with comparably-sized V8 protease fragments of H1. IgM antibodies to H1 in the other patients were directed against entirely different epitopes which were preserved in V8 protease digests of H1. In serial studies of three patients during different phase of their SLE, the level of antibodies against the 18 kD and 20 kD histone H1 fragments varied in parallel with the level of anti-ssDNA antibodies in one and varied inversely in the other two. The data suggest that a significant proportion of autoantibodies to histone H1 are directed at a limited number of epitopes localized to H1 fragments containing DNA-binding sites.     

Characterization and specificity of anti-endothelial cell membrane antibodies and their relationship to thrombosis in primary antiphospholipid syndrome (APS).

Immunoblotting was used to detect antibodies reacting with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), fibroblasts and a T lymphoma line HUT78 in 18 patients with anticardiolipin antibodies (ACA) (14 of whom had had a thrombotic event), 11 patients with a recent myocardial infarction and 17 controls. Multiple membrane-specific antibodies to HUVEC were found in 10 of the patients with ACA (28 bands) and in nine of the patients with thromboses (27 bands) in contrast to only three of the patients with myocardial infarction (four bands) and one control (one band). The most frequently recognized HUVEC membrane epitopes were at 33 kD (four sera), 61-63 kD (five sera) and 76-79 kD (four sera). Although cross-reactivity with fibroblast and/or HUT78 membranes was seen at 33 kD, binding at 61-63 kD and 76-79 kD was specific for endothelial membranes. Although no correlations with the presence and titre of ACA were seen, HUVEC membrane-specific antibodies showed a correlation with venous thrombotic events.     

A critical study of the IgG subclasses of Rh anti-D antibodies formed in pregnancy and in immunized volunteers

The IgG subclasses of Rh anti-D antibodies were investigated in a hundred anti-Rh sera using IgG subclass-specific antisera. Antibody activity was found to be entirely restricted to the IgG1 and IgG3 subclasses. Immunized volunteers tended to produce both IgG1 and IgG3 antibodies whereas antibodies from incompatible pregnancies were more often composed of only one subclass.It is suggested that some supposedly subclass `specific'' antiglobulin sera that have been standardized using weakly coated test red blood cells may show cross-reaction with more strongly coated cells and this may lead to the erroneous assumption that certain high titre anti-Rh sera also contain IgG2 and IgG4 antibodies.