MHC gene products and anticardiolipin antibodies in systemic lupus erythematosus results of a multicenter study. SLE Study Group.

Anticardiolipin antibodies (aCL) and HLA-DR antigens were determined in 314 central European patients with systemic lupus erythematosus (SLE). Both HLA-DR4 and DR7 were increased in aCL-positive patients, and aCL were significantly associated with DRw53. The association between DRw53 and aCL was also apparent in those 17 patients with SLE and the anticardiolipin syndrome. There was no association between aCL and HLA-DQ or C4 alleles in SLE.

Antibodies against p24 of HIV-1 in patients with systemic lupus erythematosus?

The involvement of retroviruses in the etiopathogenesis of autoimmunity is discussed. Recently antibodies against p24 of HIV-1 were described in patients from Texas with SLE. Therefore we investigated the presence of these antibodies in our SLE collective (German caucasians and blacks from Michigan) employing ELISA and Western blot. No anti-p24 reactivity was observed in our SLE patients in Western blots, suggesting that there may be ethnological or regional differences between our patients those from Texas.

The genetic basis of Ro and La antibody formation in systemic lupus erythematosus. Results of a multicenter study. The SLE Study Group.

Antibodies against Ro and La, including recombinant La and recombinant 60 kD-Ro, were determined by counter immunoelectrophoresis and ELISA in over 300 central European systemic lupus erythematosus (SLE) patients. The presence of both Ro and La antibodies was strongly associated with the MHC haplotype B8-C4AQ0-DR3-DQ2, the association being strongest for DR3. After exclusion of all B8-DR3 positive patients only DR3 positive patients still showed an increased incidence of Ro and La antibodies, suggesting DR3 as the primary association factor. High titers of La antibody, but not of 60 kD-Ro antibody, were also significantly associated with the presence of DR3. Other DR and DQ antigens or heterozygous DQ combinations were not significantly associated with Ro and La antibodies.

Hetero- and homozygosity of MHC class II gene products in systemic lupus erythematosus. The Members of the Deutsche Multizentrische SLE-Studie.

An analysis of HLA class II antigens in 356 white patients with systemic lupus erythematosus (SLE) showed that all HLA-DR and -DQ homozygous and heterozygous combinations appear with frequencies expected from the observed gene frequencies. HLA-DR2 and HLA-DR3 gene frequencies were both increased in SLE, as were the odds ratios of all DR2 and DR3 hetero- and homozygous combinations. HLA-DR2/C4AQ0 heterozygotes were also not increased over expected values. Therefore, gene complementation at MHC loci does not contribute to susceptibility to SLE, but rather one or two MHC allele(s) in linkage with HLA-DR2 and HLA-DR3.

[Plasma nucleic acids and their possible pathophysiologic significance in systemic lupus erythematosus]. / Plasmanukleinsäuren und ihre mögliche pathophysiologische Bedeutung beim systemischen Lupus erythematodes.

The discussion of the possible pathophysiological role of plasma and/or serum nucleic acids from patients suffering from systemic lupus erythematosus is nearly identical with the still unsolved question regarding the antigen, i.e., the possible autoantigen-inducing antibodies against native double-strand DNA (dsDNA). This question is of special interest, since native dsDNA per se is not immunogenic. As repeatedly demonstrated, circulating antibodies of the isotype IgG against dsDNA can be correlated with the disease activity and, particularly, with renal involvement. Antibodies against native dsDNA were isolated from affected organs from SLE patients. The analysis of circulating immune complexes revealed dsDNA, as well as antibodies against dsDNA as complex components. DNA-anti-dsDNA serum immune complexes could also be demonstrated in patients with a so-called seronegative SLE, i.e., in patients not showing any free antibodies against native dsDNA in their sera. Furthermore, the involvement of antibodies against native dsDNA in pathogenic mechanisms of SLE patients becomes particularly evident from animal models. Regarding the induction of dsDNA antibodies, it is of special interest that in animal models such as the MLR/lpr mouse, anti-dsDNA antibodies are rather antigen-selected than they are a consequence of a "random" polyclonal B cell stimulation. Likewise, by the demonstration of somatic mutations of clonal human IgG-anti-dsDNA antibodies from SLE patients it has recently been possible to prove that these autoantibodies are also most probably antigen-selected.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of antineutrophil cytoplasm antibodies, anticardiolipin antibodies, von Willebrand factor antigen, and fibronectin for the diagnosis of systemic vasculitis.

Autoantibodies directed against cytoplasmic components of neutrophil granulocytes and monocytes (c-ANCA) are a disease specific marker for Wegner's granulomatosis (WG). Autoantibodies against cardiolipin (aCl) are specific for a subgroup of autoimmune disorders, which can also be associated with systemic vasculitis. Fibronectin (Fn) and von Willebrand factor antigen (vWfAg) are produced by blood vessel endothelial cells in response to injury. We tested sera of 61 patients with various types of systemic vasculitides, sera of 13 patients with retinal vasculitis, and sera of 199 patients with rheumatic diseases for c-ANCA, aCl, Fn, and vWfAg. c-ANCA was positive in 14/17 patients with WG, and 2/4 with polyarteritis nodosa (PAN). No serum from healthy donors or patients with other vasculitic or rheumatic diseases was positive for c-ANCA. Moreover, we found aCl, Fn, and vWfAg significantly elevated in almost all patients with vasculitic syndromes. Therefore, we consider c-ANCA a marker, specific for the diagnosis of WG or PAN, whereas aCl, Fn, and vWfAg are nonspecific but sensitive markers of vascular damage.

Pathogenesis of SLE: immunopathology in man.

Antibodies against native DNA are not only a disease-specific marker for systemic lupus erythematosus (SLE); in addition, there is good direct evidence that these antibodies also play a major part in pathogenic mechanisms leading to systemic and organ-specific disease manifestations. The origin of anti-dsDNA antibodies is still poorly understood, especially as dsDNA per se is not immunogenic. As recently shown, evidence is now accumulating that anti-dsDNA antibodies are not germline-encoded but antigen-driven, as demonstrated by the establishment of human anti-dsDNA antibody clones from SLE patients and sequence analysis. In sera of SLE patients there is an elevated level of nucleic acids, which indicates that defective clearance mechanisms for nucleic acids are present. The question as to whether these nucleic acids could serve as an antigen has been recently addressed by studies of plasma nucleic acids isolated from circulating immune complexes from SLE patients. These studies indicate that plasma nucleic acids in SLE patients have structures of amino acid sequences which have a striking homology with the gag-pol overlap region of HIV-1. Whether these nucleic acids play a role in the pathogenesis of SLE, indicating the involvement of a retrovirus in the pathogenesis, or whether they rather reflect an amino acid homology with an endogenous human retrovirus family is not yet known.

Managing change. A step-by-step process helps sponsors implement change smoothly.

During the past six years the Catholic Health Association (CHA) has developed and modified a process to help leaders evaluate and implement merger, cosponsorship, and sponsorship transfer decisions. CHA's highest priority in these efforts has been to keep Catholic healthcare facilities under Catholic sponsorship, control, and management. Proposals to change sponsorship arrangements usually originate with sponsoring institutes, whereas local boards generally initiate merger proposals. In either case, it is critical that all interested parties--such as sponsors, boards, administrators, medical staff, employees, and the local Church--be involved in the decision-making process at some point. Once leaders have decided on a course of action, they should appoint a task force to implement the proposal. The board, administration, and medical staff will all have important roles to play in the implementation process. Another important step is to establish criteria for evaluating candidates for a proposed merger or sponsorship transfer. Leaders should ensure that people affected by the transaction have an opportunity to give input and to grieve their loss. After leaders have selected a candidate, they must negotiate the details of the agreement and take the necessary legal steps to complete the transaction. It is imperative that a facility secure outside legal counsel to help it through this stage.

Circulating immune complexes in HIV-infected persons.

Circulating immune complexes are part of normal immune defense mechanisms and, therefore, present in various infectious--bacterial and viral--diseases. On the other hand, they are obviously involved in pathogenic mechanisms, e.g., autoimmune diseases or different forms of malignancies. Both autoimmune and infectious features are recorded in the acquired immunodeficiency syndrome. Thus, elevated levels of antigen-antibody complexes in HIV-infected persons had to be expected, and they were in fact demonstrated by several authors. In a cohort study, it was additionally shown that circulating immune complexes are of prognostic relevance. After an introduction concerning the physiological and pathophysiological role of circulating immune complexes in general, their involvement in the course of HIV infections is presented and discussed. In addition, there is a critical review of the most commonly applied assay systems for the detection and quantification of circulating immune complexes.

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1.

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.

Antibody binding of macromolecular DNA and RNA in the plasma of SLE patients.

Plasmapheresis fluids from 20 patients with clinically active SLE, from three patients with Waldenstrom's disease, from three patients with rheumatoid arthritis, two patients with myasthenia gravis and other diseases including active systemic disorders were precipitated using polyethylene glycol 6000 (PEG). By applying ethidium bromide staining, plasma nucleic acids (PNA) could be demonstrated in PEG-precipitates of SLE patients exclusively. Purified immunoglobulins of SLE plasma precipitates were shown to form antigen-antibody complexes with PNA as demonstrated by electronmicroscopy. Further characterization of PNA by agarose gel electrophoresis revealed a molecular weight up to 20 kbp. Cesium chloride buoyant density gradients showed non-homogeneous molecules, excluding pure microbial origin. In spite of RNase digestion, the PNA contained RNA with 30-70% riboguanosine as shown by nucleoside analysis. The high amount of guanosine-rich RNA was further supported by similarities between PNA and polyriboguanylic acid in hyperchrome shifting due to thermic denaturation. HPLC analysis showed a molecular weight of ribonucleic acids of more than 60 b thus excluding mere oligonucleotides. In contrast to B-type dsDNA, PNA from SLE patients were immunogenic. Antibodies against PNA could be induced in rabbits by subcutaneous injection. The antisera thus obtained showed crossreactivity with polyriboguanylic acid and dsDNA preparations.

Are retroviruses involved in the pathogenesis of SLE? Evidence demonstrated by molecular analysis of nucleic acids from SLE patients' plasma.

High molecular weight DNA of up to 20 kbp and, additionally, an RNase-insensitive RNA of more than 60 b were isolated from plasmapheresis fluids taken from patients with active systemic lupus erythematosus (SLE). Similar nucleic acids could not be demonstrated in the plasma samples from patients with Waldenstroem's disease, rheumatoid arthritis, myasthenia gravis, and other diseases including active systemic disorders. The purified nucleic acids were analyzed in several ways; they proved to be immunogenic by inducing polyclonal and monoclonal antibodies to natural DNA as well as to synthetic polynucleotides (e.g. polyguanylic acid) after injection into experimental animals (rabbits or mice respectively). Biochemical and molecular cloning analysis of the DNA revealed features like high levels of CpG-dinucleotides, usually not observed in common human DNA. A possible exogenous origin was substantiated by comparative sequence studies of cloned plasma DNA, which showed homologies to human retroviruses, e.g. PL1 (85%/60 b) and the sequences of the gag and pol genes of human immunodeficiency virus type I (85%/157 b). Experiments applying isolated plasma nucleic acids in transfection experiments showed the induction of morphological changes in an EBV-immortalized B-cell line drawn from a healthy human donor, such as vacuolization and syncitia formation. Northern blot analysis demonstrated, exclusively in the transfected cell line, the expression of mRNA homologous to the cloned plasma DNA. Using this clone as a probe, homologous sequences could be demonstrated by Northern blot analysis in EBV-immortalized cell lines from SLE patients only and, by means of DNA amplification, in peripheral blood lymphocytes from SLE and AIDS patients.(ABSTRACT TRUNCATED AT 250 WORDS)

C3-containing serum immune complexes in patients with systemic lupus erythematosus: correlation to disease activity and comparison with other rheumatic diseases.

Although testing for circulating immune complexes (CIC) is regarded as a useful, complementary, laboratory parameter in the differential diagnosis and management of immune complex-induced vasculitis syndromes, there is still an uncertainty with regard to assay systems used for the demonstrated of soluble immune complexes. This is partly due to difficulties in the reproducibility, handling and principle limitations of available test systems for the assessment of soluble immune complexes in body fluids. In the present communication a modification of the anti-C3 test for the determination of CIC was developed using nitrocellulose as a solid phase matrix. IgG-, IgA- and IgM-containing CIC were determined and quantified using standard immune complex preparations. When 39 sera of SLE patients, 12 sera of patients with vasculitis syndromes, 10 sera of rheumatoid arthritis patients and 11 sera of patients with ankylosing spondylitis were tested, predominantly IgG-containing CIC could be demonstrated. Only in SLE patients was a significant amount of other immunoglobulin isotypes detected in CIC. In these patients a significant difference of IgG-containing CIC levels was found with regard to patients with high and low disease activity (P less than 0.0001). A significant correlation was also established between IgG-containing CIC and anti-dsDNA antibodies (P less than 0.001). In a longitudinal study the isotypes in the isolated CIC were found to be constant.

IGM-containing immune complexes and antiphospholipid antibodies in patients with Sneddon's syndrome.

We report three patients with a Sneddon syndrome in whom predominantly small (500-900 kD) IgM-containing serum immune complexes were detectable. Furthermore, antiphospholipid antibodies and increased von Willebrand factor antigen were found in the sera of two cases. Especially the data demonstrating small circulating immune complex as suggest that Sneddon's syndrome, a rare vasculitis disorder, might immunologically be characterized by circulating IgM-containing immune complexes which, in addition, could play a role in the pathogenesis of this disease entity. The elevated antiphospholipid antibodies as well as the increased von Willebrand factor antigen in the sera of the investigated patients have to be considered as nonspecific vasculitis-associated phenomena.

CEA-containing immune complexes in sera of patients with colorectal and breast cancer--analysis of complexed immunoglobulin classes.

A sandwich enzyme immunoassay was developed to detect circulating immune complexes containing carcinoembryonic antigen (CEA) and immunoglobulin (Ig) G, IgA, or IgM using a nitrocellulose-bound anti-CEA antibody as the solid phase reagent. Elevated levels of CEA-containing circulating immune complexes (CEA-IC) were found in 15.4% of 117 sera from patients with colorectal cancer in a postsurgery follow-up study. Also in 24.5% of 102 sera from patients with breast cancer in different states of disease CEA-IC were found. The predominant Ig determined in CEA-IC of colorectal cancer patients was IgA, followed by IgG and IgM, whereas IgG and IgM were the most frequent Igs in CEA-IC of breast cancer patients. Elevated CEA levels were found in 12.0% of the colorectal cancer patients and in 25.4% of sera from breast cancer patients. No significance for the coincidence of elevated CEA levels and CEA-IC was recorded in all patients sera tested. In sera of patients with disease recurrence, however, both parameters were shown to be elevated (CEA 80.7% and CEA-IC 42.3%). The data presented indicate the detection of CEA-IC as an additional parameter for the identification of patients at increased risk for disease recurrence.

Determination of complement activating circulating immune complexes and an example for antigen specific immune complex assays.

For the screening of complement activating circulating immune complexes a new fluorescence linked immunosorbent assay was developed. Anti-human C3 or anti-human C4 F(ab')2 fragments were coupled to a nitrocellulose matrix. Nitrocellulose pieces of definite size covered with anti-C3 and anti-C4 were incubated with serum samples or standards for 15 min followed by a reaction with a fluorescence labelled anti-human IgG or anti-human-IgM. The nitrocellulose disks were then washed and the remaining fluorescence was read by a solid-phase fluorometer. The method was standardized by a WHO Tetanus toxoid anti-Tetanus immune complex reference substandard. (This standard and further controls were used to prove the reproducibility of the system.) Patients suffering from chronic inflammatory diseases were compared to healthy controls. The best discrimination between patients and controls was demonstrated by the determination of C3-IgG aggregates, followed by C3-IgM, C4-IgG, and C4-IgM aggregates. The easy performance, the stability of necessary chemical substances, the reliable standardization by a reference standard, and the clear-cut differences between patients and healthy control groups proved this test to be suitable for routine purposes. Furthermore, it could be demonstrated by means of an artificial model immune complex that this system can be expanded--by slight modifications--to antigen-specific CIC-assays. A CEA-anti-CEA CIC test is described as an example of an antigen specific CIC test not limited to complement activating CIC, and the data of 127 patients with colorectal carcinoma are given.