Specific detection of circulating DNA:anti-DNA immune complexes in human systemic lupus erythematosus sera using murine monoclonal anti-DNA antibody.

The presence of DNA-anti-DNA immune complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by a new solid phase radioimmunoassay (RIA). This assay used murine monoclonal anti-double stranded DNA (dsDNA) antibody to recognize DNA present in the complexes and 125I-rabbit anti-human gamma globulin as a tracer. DNA-anti-DNA immune complexes were found in certain SLE sera but not in sera from patients with other immune complex diseases and from healthy blood donors. The presence of circulating DNA-anti-DNA complexes was associated with low C4 levels. It was not related to the presence of immune complexes detected by the polyethylene glycol assay suggesting either that the assay did not detect all DNA-anti-DNA complexes or that other antigen-antibody systems constitute the major immune complex components in SLE sera. The clinical significance of circulating DNA-anti-DNA complexes in SLE sera as well as the potential use of this solid phase RIA using various monoclonal antibodies to detect specific antigen-antibody systems is discussed.     

Antigen detection in immune complexes by a modified staphylococci binding assay and Western blot analysis

The combination of a modified Staphylococci binding assay for immune complexes and Western blot analysis is described for the isolation and detection of antigen in immune complexes from human sera. The strategy of the procedure is to preclear immune complexes from other serum components by sequential polyethylene glycol precipitation and incubation with insoluble protein A under conditions in which immune complexes are preferentially bound. Immune complexes are eluted from protein A in sodium dodecyl sulfate buffer and dissociated by acrylamide electrophoresis. Resolved proteins are then transblotted to nitrocellulose, and antigen is detected with specific antibody. Immune complexes were prepared in vitro with an antigen (keyhole limpet hemocyanin) that focuses well on electrophoresis and for which a potent immunologic probe (antibody) was available. In this system, antigen could be detected when immune complexes were present in sera in concentrations as low as 20 micrograms aggregated-IgG eq/ml, regardless of antigen-antibody ratio. We demonstrate the detection of horse globulin in immune complexes from a patient with acute serum sickness and hepatitis B virus antigen in complexes from a patient with vasculitis.     

A simple quantitative assay of circulating immune complexes by laser nephelometry, using a rabbit igg antibody against human aggregated igg

Circulating antigen-antibody complexes were detected by measuring agglutination of complexes with rabbit antisera against heat-aggregated human IgG by means of laser nephelometry. Rabbit antisera were obtained by immunization with heat-aggregated human IgG after intravenous injection of a large amount of native human IgG. The antisera were observed to be almost specific to conformationally altered human IgG by immunodiffusion. Even if the antibody activity to native IgG did occur in the antisera, the native IgG in test sera was in excess of antigen in the assay system employed for the detection of immune complexes. In conclusion, the minor antibody activity against native IgG does not interfere with the assay of immune complexes by laser nephelometry. The clinical applications demonstrated the advantage of the method.     

Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.     

Complement-dependent cytotoxic antibodies to human T lymphotropic virus type I (HTLV-I)-infected cells in the sera of HTLV-I-infected individuals

To investigate whether HTLV-I induces the development of complement-dependent cytotoxic antibodies in humans, sera of asymptomatic HTLV-I carriers and of patients suffering from tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) or adult T cell leukaemia (ATL) were used in a cytotoxicity assay against a panel of target cells. This panel included uninfected cell lines (CEM, Jurkat, Molt and H9), cell lines chronically infected with HTLV-I (MT2, MT4, C91PL and HUT102), as well as lines H36 (H9 infected with HTLV-I), H9-IIIB (H9 infected with HIV_IIIB) and H9-MN (H9 infected with HIV_MN). HTLV-I⁺ sera induced lysis of H36 and of lines expressing HTLV-I antigens in the presence of rabbit complement, but did not lyse cells in presence of human complement. The HTLV-I⁺ sera also failed to lyse the HTLV-I⁻ lines and H9 cells, suggesting that lysis was specific for HTLV-I. H36 cell lysis was prevented by IgG depletion of the sera and by dialysis of rabbit complement against EGTA or EDTA. Rabbit complement-dependent cytotoxic antibodies were present in the sera of 14/14 HTLV-I-infected individuals; the highest titres were predominantly found in the sera of the TSP/HAM patients. Such antibodies were also detected in 5/5 individuals coinfected with HIV-1 and HTLV-I, although no cytotoxic antibody could be found against HIV-infected cells. Vice versa, sera of HIV-1-infected individuals did not exert a lytic effect in the presence of complement (of human or rabbit origin) against HIV-1- or HTLV-I-infected cells. Incubation of the sera of four HTLV-I-infected patients with HTLV-I env-specific synthetic peptides demonstrated that some of the complement-dependent cytotoxic antibodies recognized epitopes located on gp46 between amino acids 190 and 209. There is no correlation of rabbit complement-dependent cytotoxic HTLV-I antibodies with the development of disease.     

Immune complex detection by immunofluorescence on polymorphonuclear leucocytes.

Polymorphonuclear leucocytes (PMN) from patients with systemic lupus erythematosus (SLE) were isolated from defibrinated and heparinized blood. In addition, PMN from a healthy donor were incubated with sera from SLE patients and with sera containing artificially prepared immune complexes of hepatitis B surface antigen (HBsAg) and human anti-HBsAg immunoglobulin (anti-HBs) with well defined variations of the antigen/antibody ratio. To one group of blood samples, 5 mM monoiodine acetic acid (MIAA) was added to block in vitro phagocytosis. The Pmn were examined for the presence of IgG, IgM, and HBsAg by the immunofluorescence technique. PMN from defibrinated blood of SLE patients showed in up to 80% immunoglobulin (Ig)-inclusions. However, addition of 5 mM MIAA reduced the number of Ig-containing PMN to at most 40%, which levels were equal to numbers found in specimens from heparinized blood. Addition of 5 mM MIAA to heparinized blood did not reduce the number of PMN with Ig inclusions. Normal donor PMN isolated from defibrinated, heparinized, and EDT blood showed equal amounts of Ig inclusions after incubation with SLE sera, but none when MIAA had been added. In PMN incubated with HBsAg-anti HBs immune complexes with an antigen antibody ratio between 5 and 0-2, both HBsAg and IgG could be detected. It is concluded that Ig inclusions in PMN from heparinized blood from SLE patients are due to in vivo phagocytosis, presumably of circulating immune complexes. In vitro phagocytosis of Ig from SLE sera by normal donor PMN also suggests the presence of immune complexes. Dependent on the antigen-antibody ratio, artificial HBsAg/anti-HBs immune complexes can be detected by in vitro phagocytosis by PM.     

A protein A-binding, polyethylene glycol precipitation-based immunoradiometric assay. Application to the detection of immune complexes and C3 in human sera and of private antigens in cross-reacting parasite extracts

An immunoradiometric assay, based on the precipitation of antigen-antibody complexes by polyethylene glycol (PEG) and on the subsequent binding of PEG-soluble radiolabelled staphylococcal protein A to the PEG-insoluble complexes, is described. The assay can be applied to the detection of naturally occurring, circulating immune complexes, and of complexes artificially created by mixing antigen and antibody solutions, which makes it of potential use for the detection of either antigen or antibody in several situations. Pre-treatment of the antibody-containing sera with 3% PEG greatly reduced the background values and increased the sensitivity of the assay. The assay was also applied to the detection and isolation of Leishmania donovani antigens that did not cross-react with antigens of the related parasite Trypanosoma cruzi (private antigens) and private antigens of insect-derived metacyclic trypomastigotes of T. cruzi in relation to culture-derived metacyclic trypomastigotes of T. cruzi. A simple and extremely effective procedure for washing precipitates with just one centrifugation is also described.     

Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody.

The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the ¹²⁵I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, ¹²⁵I-labelled Clq BA and Raji-cell radioimmunoassay (RIA).

Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.

     

Natural killer activity and antibody-dependent cellular cytotoxicity in patients with adult T-cell leukemia

Natural killer (NK) activity of peripheral mononuclear cells (PMNC) from patients with adult T-cell leukemia (ATL), anti-HTLV-I antibody positive healthy carriers, and anti-HTLV-I antibody negative healthy persons (Ab-negative persons) was investigated using various target cells. PMNC from patients with ATL and healthy carriers had reduced NK activity against the NK-sensitive non-HTLV-I producing target cells, compared with the controls. In contrast, PMNC from patients with ATL, healthy carriers, and Ab-negative persons did not exhibit significant NK cell lysis against HTLV-I producing cells. However, one Ab-negative person exhibited increased NK cell lysis against an HTLV-I producing target cell. The effector cells involved in this enhanced cytolysis were found to be CD3+, HNK-1+, and CD8+. HTLV-I producing cells were lysed by PMNC from Ab-negative persons in the presence of anti-HTLV-I antibody (antibody-dependent cellular cytotoxicity; ADCC). The efficiency did not show significant difference between antibodies from patients with ATL and those from healthy carriers. The ADCC was specific to HTLV-I producing cells. PMNC from one patient with ATL in remission exhibited increased ADCC in the presence of autologous serum against HTLV-I, whereas PMNC from a patient with ATL or a healthy carrier did not exhibit ADCC. These results indicated that NK and K cells influence the immunological surveillance against HTLV-I infection or leukemic cells.     

Chronic liver disease: the detection and characterization of circulating immune complexes.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.     

Immune suppression in HTLV-I carriers: a predictive sign of adult T-cell leukemia

Suppression of the cellular immune system appears to be a prerequisite for the manifestation of adult T-cell leukemia (ATL). In other words, ATL will develop when impairment of the immune system is caused by the infection of human T-lymphotropic virus type I (HTLV-I). This defect of immune surveillance against virus-infected cells may be a result of the impairment of the function of cytotoxic T-cells (CTLs) specific for the HTLV-I-infected cells. The manifestation of ATL could be predicted by examining the function of CTLs in HTLV-I carriers. A new strategy of prevention and therapy for ATL would include an attempt to restore and fortify the CTL function of the host.     

Inhibition of antibody-dependent cellular cytotoxicity by artificial immune complexes and pathological sera.

Detection of immune complexes by inhibition of antibody-dependent cellular cytotoxicity (ADCC) is based on the principle that soluble complexes can compete with target cell-bound antibody for receptors (FcR) on cytotoxic lymphocytes. The objective of this study was to define a cytotoxicity system for the determination of soluble immune complexes in the sera of patients with inflammatory bowel disease (IBD). For this purpose, the conditions under which soluble complexes of rat serum albumin (RSA) and rabbit anti-RSA inhibited human K-cell mediated lysis of sensitized Chang cells were examined, on the assumption that the behaviour in the system of circulating immune complexes putatively present in inflammatory bowel disease, is similar to that of artificial immune complexes. Inhibition of ADCC by a standard amount of artificial complex in different normal human sera was relatively uniform provided that the final concentration of the latter did not exceed 10% of the culture medium. In the absence of extraneous complexes, the effect of both normal and IBD sera on ADCC varied widely. Differential inhibition of ADCC by sera from patients with IBD and normal subjects was thus expressed as a function of ADCC in a standard batch of foetal bovine serum (FBS). Under these conditions differences between pathological (n = 51) and normal (n = 52) sera were highly significant (P less than 0.001), which could not be explained by the presence in the patients' sera of HL-A antibodies reactive with the effector cells, nor by a deficit in nutritional support of ADCC. The absence of a correlation between inhibition of ADCC and total serum IgG or IgM inferred that inhibition was attributable to immune complexes in the IBD sera. The limitations of this assay for assessment of the incidence of immune complexes in pathological sera are discussed.     

Complement-mediated inhibition of immune precipitation in patients with immune complex diseases

The ability of human sera to prevent the precipitation of antigen-antibody complexes has been investigated. The early complement components including C3 are required for optimal prevention of immune precipitation, whereas the later components are not required. The sera of 36 of 75 patients with seropositive rheumatoid arthritis (RA), 14 of 32 with SLE and four of 17 with glomerulonephritis exhibited reduced capacities to prevent immune precipitation. In contrast sera from patients with seronegative RA, ankylosing spondylitis, psoriatic arthritis or degenerative joint disease were normal in this respect. In SLE and GN sera hypocomplementaemia was frequently associated but not always with failure to prevent immune precipitation, whereas only a small proportion of the patients with seropositive RA and reduced capacity to retain complexes in a soluble form were hypocomplementaemic. Thus the failure of sera to prevent the precipitation of antigen-antibody complexes is not always associated with hypocomplementaemia.     

In vitro studies of the normal human B lymphocyte receptors for preformed soluble dengue antigen-antibody complexes.

Preformed soluble dengue antigen-antibody with or without complement complexes could bind to the surface of human lymphocytes via IgG (Fc) or complement receptors respectively. It may be that these lymphocytes were B lymphocytes not T lymphocytes. Dengue antigen or antibody alone will not attach to the surface of those cells. It is emphasized that dengue antigen-antibody complexes can form in the circulation and some will bind to the surface of the B lymphocytes in vivo and play a significant role in the pathogenesis of dengue haemorrhagic fever. In addition mice could develop glomerulonephritis by injection with preformed soluble dengue antigen-antibody complexes. These mice showed proteinuria and deposits of the immune complexes in the glomeruli of the kidneys.     

Leukaemogenic mechanism of human T-cell leukaemia virus type I

Adult T-cell leukaemia (ATL) is a neoplastic disease derived from CD4(+) T-lymphocytes and etiologically associated with human T-cell leukaemia virus type I (HTLV-I). In addition to structural genes, HTLV-I encodes regulatory and accessory genes in the pX region. Among them, Tax is thought to play a central role in leukaemogenesis through its potent transforming activity. However, since Tax is a major target of the host immune system, its expression is often lost in ATL cells, indicating Tax is dispensable in the last phase of leukaemogenesis. The HTLV-I bZIP factor (HBZ), encoded on the HTLV-I minus strand, was recently shown to be expressed in all ATL cells, and to support growth of human T-cell lines. These findings suggest that HBZ is critical to ATL onset. In addition to viral factors and genetic and epigenetic changes in cellular genes, the host immune status and genetic background also function in leukaemogenesis.     

Nephelometric detection of soluble immune complexes: methodology and clinical applications.

We have developed a method for the detection of immune complexes by laser nephelometry which is simple, reproducible, suitable for automation, and generally adaptable for diagnostic testing. Light dispersion by antigen-antibody complexes in the test samples is measured after addition of polymeric buffer, which enhances the aggregation of complexes but does not significantly affect unbound immunoglobulins. The method was used to measure immune complexes formed in vitro by incubation of tetanus toxoid with serum from a rabbit previously hyperimmunized with the same antigen, and to compare the levels of immune complexes in human sera obtained from normal adults and from 37 patients with collagen vascular diseases or endocarditis. When precautions were taken to avoid interference produced by the presence of lipoproteins or by freezing of the samples, the results obtained with human sera were consistent with those expected for normal controls and for patients with conditions thought to be associated with the presence of soluble immune complexes.     

Cytogenetic implication in adult T-cell leukemia. A hypothesis of leukemogenesis

The close association between adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I) has been established. Nevertheless, the mechanism of progression of ATL by HTLV-I infection is still uncertain, because the virus contains no typical oncogene and no significant expression of the viral RNA has been generally found. I propose a model of leukemogenic process in ATL based on our cytogenetic data and molecular results in the literature. It seems that the rearrangement of some proto-oncogene and alpha-chain gene of the T-cell antigen receptor (TCR-alpha) is necessary for the development to overt ATL. A deficiency in the rearrangement of proto-oncogene to TCR-alpha may result in only a minor proliferation of abnormal lymphocytes and remain in the preleukemic state of ATL or in the HTLV-I carrier state.     

Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.     

Identification of HTLV p19 specific natural human antibodies by competition with monoclonal antibody

A competitive binding assay using a monoclonal antibody to the human T-cell lymphoma/leukemia virus (HTLV) p19 was developed for use in detecting natural antibodies to the protein in human sera. The specificity of the assay for HTLV p19 was demonstrated using a variety of antisera. While sera known to contain antibodies to HTLV p19 competed in the assay, antisera prepared against purified HTLV p24, the major core protein of the virus, or against other disrupted type-C retroviruses did not. Sera of Japanese patients with adult T-cell leukemia and similar T-cell malignant lymphomas were examined by this technique for the presence of antibodies to HTLV p19. The results were compared with those obtained by a solid-phase radioimmunoassay (RIA) against disrupted HTLV. The majority of Japanese ATL patients possess natural antibodies to HTLV as shown by solid-phase RIA (88%) and also specifically to HTLV p19 (77%). Similarly, 50% of Japanese patients with similar T-cell malignant lymphomas possess HTLV antibodies by solid-phase RIA and nearly as many (42%) possess anti-p19 reactivity. Twelve and eight percent, respectively, of normal Japanese donors from the ATL endemic region possessed HTLV-specific antibody by the solid-phase RIA or competitive binding assay. Normal donors from nonendemic areas lacked antibodies to HTLV. These results extend our previous findings of natural antibodies to HTLV in Japanese patients with ATL. The finding of p19-specific antibodies in these Japanese sera, together with previous reports of natural antibodies to HTLV p24 in sera from this same geographic cluster, strengthens the association of HTLV with Japanese ATL.     

Circulating IgG complexes in primary biliary cirrhosis. A serial study in forty patients followed for two years.

Several antibodies are present in sera of patients with primary biliary cirrhosis (PBC). We have looked for evidence of antigen-antibody complexes in sera of PBC assuming that some of the antibodies may circulate complexed with an antigen. The Raji cell radioimmunoassay, which determines complement-bound immune complexes, was used to determine the levels of such complexes in serial samples of sera from forty patients with PBC followed for 2 years. Twenty-four patients (60%) were found to have significantly elevated levels of circulating complexes. In the majority they were detected from the beginning of the study and the high levels persisted. In seven patients whose sera initially had normal levels of complexes, the levels increased to become abnormal during the following year. These complexes sedimented at greater than or equal to 19S in the majority of patients studied. The mean level of C3 but not C4 was lower in patients with elevated complexes than in those with normal complexes. A significant correlation was observed between the presence of elevated complexes and the severity of the inflammatory cell infiltrate surrounding intrahepatic portal tracts and serum IgG and IgM levels. There was also a significant correlation with titres of antimitochondrial antibody, but not with the histological stage of disease or with the collagen and copper content of the liver. Although the method of detection of immune complexes is indirect and the antigen is unknown, the presence of such high levels of complexes suggests a possible role of immune complexes in the pathogenesis of PBC.