A sensitive and specific assay for the serological diagnosis of antilaminin 332 mucous membrane pemphigoid.

BACKGROUND: Antilaminin 332 mucous membrane pemphigoid (MMP) is an autoimmune subepidermal blistering disease with predominant mucosal involvement and autoantibodies against laminin 332. Malignancies have been associated with this disease; however, no standardized detection system for antilaminin 332 serum antibodies is widely available. OBJECTIVES: Development of a sensitive and specific assay for the detection of antilaminin 332 antibodies. METHODS: An indirect immunofluorescence (IF) assay using recombinant laminin 332 was developed and probed with a large number of antilaminin 332 MMP patient sera (n = 93), as well as sera from patients with antilaminin 332-negative MMP (n = 153), bullous pemphigoid (n = 20), pemphigus vulgaris (n = 20) and noninflammatory dermatoses (n = 22), and healthy blood donors (n = 100). RESULTS: In the novel IF assay, sensitivities with the laminin 332 heterotrimer and the individual α3, ß3 and γ2 chains were 77%, 43%, 41% and 13%, respectively, with specificities of 100% for each substrate. The sensitivity for the heterotrimer increased when an anti-IgG4 enriched antitotal IgG conjugate was applied. Antilaminin 332 reactivity paralleled disease activity and was associated with malignancies in 25% of patients with antilaminin 332 MMP. CONCLUSIONS: The novel IF-based assay will facilitate the serological diagnosis of antilaminin 332 MMP and may help to identify patients at risk of a malignancy.

The avidity of PR3-ANCA in patients with granulomatosis with polyangiitis during follow-up.

The objective of this study is to investigate whether the avidity of proteinase-3-anti-neutrophil cytoplasmic antibody (PR3-ANCA) changes during follow-up in different subgroups of patients with granulomatosis with polyangiitis (GPA). We selected 10 patients with renal relapsing GPA, 10 patients with renal non-relapsing GPA and 10 patients with non-renal relapsing GPA. In all patients, an ANCA rise occurred during remission. The avidity was measured using a chaotropic approach at the time of an ANCA rise and at the time of a relapse in relapsing patients or time-matched during remission in non-relapsing patients. No difference was observed in the avidity at the ANCA rise between renal relapsing patients [26·2% (15·5-47·5)], renal patients without a relapse [39·6% (21·2-63·4)] and non-renal relapsing patients [34·2% (21·6-59·5)]. In renal relapsing patients, the avidity increased significantly from the moment of the ANCA rise to the relapse [difference 6·4% (0·0-17·1), P = 0·0273]. The avidity did not increase after an ANCA rise in renal non-relapsing patients [difference 3·5 (-6·0 to 10·1), P = 0·6250] or in non-renal relapsing patients [difference -3·1% (-8·0 to 5·0), P = 0·5703]. The avidity of PR3-ANCA increases after an ANCA rise during follow-up in renal relapsing patients, but not after an ANCA rise in renal patients who remain in remission or in non-renal relapsing patients.

Epitope mapping of BP230 leading to a novel enzyme-linked immunosorbent assay for autoantibodies in bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive. OBJECTIVES: To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. METHODS: Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230-C3 fragment comprising the C-terminal portion (amino acids 2326-2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). RESULTS: Among all the fragments, BP230-C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230-C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti-BP180-NC16A-4X ELISA alone. CONCLUSIONS: Recombinant BP230-C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.

A novel enzyme-linked immunosorbent assay using a mixture of human native and recombinant proteinase-3 significantly improves the diagnostic potential for antineutrophil cytoplasmic antibody-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) with a C-ANCA or P-ANCA pattern are detected in ANCA-associated vasculitis (AAV). While in most patients with AAV a C-ANCA pattern is due to reactivity with proteinase-3 (PR3)-ANCA, some C-ANCA-positive sera do not react with PR3. OBJECTIVE: The development and evaluation of a direct enzyme-linked immunosorbent assay (ELISA) for PR3-ANCA with increased sensitivity. METHODS: A mixture of human native (hn) and human recombinant (hr) PR3 was used as antigen coating. The resulting ELISA (anti-PR3-hn-hr) was compared with ELISAs using directly coated hn-PR3 or hr-PR3, as well as with a hn-PR3 capture ELISA. Assay characteristics were determined in patients with AAV (n = 248), with special attention for those patients with C-ANCA (n = 132), as well as disease controls (n = 585) and healthy controls (n = 429). Additionally, for prediction of relapses serial samples of 46 patients with PR3-AAV were analysed. RESULTS: At a predefined specificity of 99% both ELISAs containing hr-PR3 revealed a substantial increase in sensitivity. For the prediction of relapses by rises in PR3-ANCA titres the capture ELISA was most optimal (odds ratio 12.5). With an odds ratio of 8.9 the novel anti-PR3-hn-hr ELISA was second best. CONCLUSIONS: Owing to the very high sensitivity of the novel anti-PR3-hn-hr ELISA for the detection of PR3-ANCA in C-ANCA-positive samples of patients with AAV this assay has an excellent diagnostic performance. This feature is combined with a good predictability of clinical relapses in patients with PR3-AAV. These characteristics challenge the dogma that, for detection of PR3-ANCA, capture ELISAs are superior for diagnosis and follow-up.

Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and clinical characteristics.

BACKGROUND: Antibodies to soluble liver antigen/liver pancreas (SLA/LP) are specific markers of autoimmune hepatitis. Their target antigen has recently been cloned. AIMS: To establish standardised immunoassays using the recombinant antigen, and to assess the frequency and significance of seropositivity in patients from different countries. METHODS: An enzyme linked immunoassay was developed using purified recombinant antigen and validated by testing sera from 200 healthy blood donors and 1026 patients with various liver and non-liver diseases. The assay was then applied to 454 sera from 419 patients with autoimmune hepatitis from different countries. All sera were also tested by inhibition immunoassay and western blot. RESULTS: Antibodies were reliably detected by the recombinant immunoassay and occurred exclusively in patients with autoimmune liver disease. Twenty three of 149 patients from the USA (15%), 23/132 from Brazil (17%), 21/108 from Germany (19%), and 2/30 from Japan (7%) were seropositive. Clinical features at presentation were similar between seropositive and seronegative patients. However, relapse after corticosteroid withdrawal or during maintenance therapy occurred more commonly in seropositive patients. CONCLUSIONS: Antibodies to SLA/LP can be reliably detected by these standardised immunoassays based on recombinant antigen. Antibodies to SLA/LP occur with similar frequencies in different geographical regions, races, and age groups, and are of exquisite diagnostic specificity. Whether SLA/LP positive patients represent a clinically distinct subgroup remains to be determined; relapse during treatment reduction appeared to be more common in the SLA/LP group.

Impact of viral and bacterial infectious burden on long-term prognosis in patients with coronary artery disease.

BACKGROUND: The number of infectious pathogens to which an individual has been exposed (infectious burden) may correlate with coronary artery disease (CAD). In a prospective study, we evaluated the effect of 8 pathogens and the aggregate pathogen burden on the risk for future fatal cardiac events among patients with angiographically documented CAD. Methods and Results-In 1018 patients, IgG or IgA antibodies to herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, Haemophilus influenzae, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Helicobacter pylori were determined. Moreover, highly sensitive C-reactive protein was measured. Follow-up information on cardiovascular events was obtained (mean 3.1 years, maximum 4.3 years). Seropositivities to Epstein-Barr virus (P=0.001), H pylori (P=0.002), and herpes simplex virus type 2 (P=0.045) were independently associated with the future risk of cardiovascular death. An increasing number for pathogen burden was significantly predictive of the long-term prognosis (P<0.0001). Infectious burden divided into 0 to 3, 4 or 5, and 6 to 8 seropositivities was associated with an increasing mortality of 3.7%, 7.2%, and 12.6%, respectively. Patients seropositive to >5 pathogens compared with those seropositive to <4 pathogens had a 5.1 (1.4 to 18.3) higher risk of future cardiac death. This result was mainly driven by the pathogen burden of seropositivities to Herpesviridae (P<0.0001). The prognostic impact of total or viral pathogen burden was independent of the C-reactive protein level. CONCLUSIONS: These results support the hypothesis that the number of infectious pathogens to which an individual has been exposed independently contributes to the long-term prognosis in patients with documented CAD.

A population of autoantibodies against a centromere-associated protein A major epitope motif cross-reacts with related cryptic epitopes on other nuclear autoantigens and on the Epstein-Barr nuclear antigen 1.

Autoimmune diseases arise from a host's immune response against self-antigens. The triggering events ultimately resulting in such a break of tolerance are largely unknown. It is also not known why certain molecular structures become autoantigenic. The hypothesis has long been proposed that autoimmune diseases arise from molecular mimicry followed by an epitope spreading mechanism. Recently we have shown that the anti-centromere-associated protein A (CENP-A) immune response is directed against an autoantigenic motif, G/A-P-R/S-R-R, that occurs three times in the N-terminal amino acids of CENP-A. In the present study we used mutational analyses with immobilized oligopeptide arrays to identify the amino acids in this motif that are responsible for antibody binding. In particular, we found that surprisingly mimotopes of this motif are present in a vast number of autoantigens and in the Epstein-Barr nuclear antigen 1. With affinity-purified antibodies we show that the antibodies against this motif are polyclonal and cross-react with several autoantigens. However, in these autoantigens this motif often represents a cryptic epitope explaining the obvious conflict between our results and the known high specificity of autoantibodies. The presence of such an ubiquitous structure on autoantigens suggests a novel peptide-driven mechanism for the evolution of autoantibodies.

IgM/IgA nephropathy in callitrichids: antigen studies.

Renal tissues of callitrichids with IgM nephropathy were immunohistochemically examined for the participation of IgA in pathogenesis. In 58 histopathologically nephropathy-positive kidneys, IgM predominated in 20 cases and IgA in 7 cases, and in 31 cases both immunoglobulins were rated to be approximately equally involved. The disease, therefore, might be described as IgM/IgA nephropathy. The renal tissues and sera were also tested for nutritional antigens or antinutritional antigen antibodies, using immunohistochemistry and Western blots (tissues) and enzyme-linked immunosorbent assay (sera). Evidences of nutritional antigens in the renal tissues were inconclusive, although circulating IgG class antibodies against cereals, milk, and egg proteins were present in quite a number of sera. Particular consideration was paid to IgA-antigliadin antibodies, which were statistically significantly associated with nephropathy as were IgA rheumatoid factors. The findings are discussed in relation to human IgA and IgM nephropathies.

Collagen synthesis in cultured aortic smooth muscle cells. Modulation by collagen lattice culture, transforming growth factor-beta 1, and epidermal growth factor.

We investigated the effects of transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the protein synthesis and production of collagen in cultured smooth muscle cells (SMCs) from the aortic media of pigs. SMCs were cultured as monolayers on plastic as well as in three-dimensional collagen lattices to gain some information about the influence of a preexisting collagenous matrix on the growth factor-induced effects. A 48-hour exposure of SMCs to TGF-beta 1 at concentrations of 5 ng/ml in the presence of 1% serum caused a marked enhancement of the production of collagen and noncollagen proteins. The rate of net collagen production by SMCs exposed to TGF-beta 1 was approximately threefold higher than that of control cells. Moreover, TGF-beta 1 specifically stimulated collagen synthesis, resulting in a greater proportion of collagen in total proteins synthesized compared with controls. The preexisting matrix of collagen lattices affects the response of SMCs to TGF-beta 1 and EGF. In monolayer cultures the collagen proportion increased twofold under the influence of TGF-beta 1, whereas in collagen lattices the specific stimulation of collagen synthesis was lower. We found that EGF enhanced TGF-beta 1-induced protein production in collagen lattices but not in monolayer cultures. In addition, the protein production by SMCs was influenced differently by EGF in these culture systems. Taken together, these data show a mutual influence of growth factors and extracellular matrix components on collagen production in SMCs, thus indicating that TGF-beta 1 may be an important pathophysiological regulator of collagen metabolism in the vessel wall.

Aortic smooth muscle cells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis.

Adult pig smooth muscle cells (SMC) were isolated from the aortic media by collagenase digestion, subcultured as monolayer, and then re-integrated into a three-dimensional network of type I collagen. The contractile state characteristic for resident arterial wall SMC changed to the synthetic, fibroblast-like state. The cells reorganized the randomly orientated collagen fibrils causing the lattice to shrink. The influence of the extracellular matrix on the ultrastructure, the proliferation, and the collagen synthesis of these SMC embedded in the collagen lattice was investigated and compared to cells cultured in monolayer. The amount of total protein and collagens synthesized by SMC embedded in lattices was lowered as compared to monolayer cultures. Whereas total protein synthesis decreased continuously during the culture period, the proportion of collagen synthesis remained at a constant level. Although cells proliferated in lattices, proliferation was clearly slowed down as compared to monolayer cultures. The ultrastructure of entrapped synthetic state SMC was comparable to that of monolayer-cultured cells. Their cytoplasm was largely filled by elements of the endoplasmic reticulum, Golgi complexes and abundant mitochondria. With prolonged culture time, electron-dense granules as well as bodies containing whorled membranes could be found in the cytoplasm. These results indicate that synthetic state SMC can exhibit differential biosynthetic activity dependent on the actual matrix environment; cells seem to be able to sense the macromolecular composition of the extracellular matrix and to modify their production of matrix components accordingly.

[Ultrastructural and proliferation studies of human, catheter atherectomy extracted plaque material]. / Ultrastruktur- und Proliferationsuntersuchungen an humanem, durch Katheteratherektomie gewonnenem Plaquematerial.

Atherectomy with a Simpson catheter enables a detailed characterization of percutaneously removed human atheromatous plaque material. Cell biological investigations (flow-cytometric analyses, cell type-specific antibodies, immunohistological labelling methods of selected extracellular matrix proteins) are aimed at a comparative characterization of excised material from primary lesions and restenoses to yield further insight into the mechanisms of plaque formation. First results on 17 specimens would like to contribute to a better understanding of the structure and composition of the extracellular matrix and their interaction with cells in atherosclerotic plaques.

Influence of collagen lattice on the metabolism of small proteoglycan II by cultured fibroblasts.

Small dermatan sulphate proteoglycan II from cultured human skin fibroblasts interacts with type I collagen in vitro and in vivo. When fibroblasts are maintained in a type I collagen lattice the proteoglycan remains exclusively within the lattice, and its association with fibrils can be demonstrated immunocytochemically. On the basis of [35S]sulphate incorporation, small proteoglycan II comprises about 80% of total proteoglycans secreted by cells in monolayer culture. In a collagen lattice, fibroblasts down-regulate its synthesis to the level of large chondroitin sulphate/dermatan sulphate and of heparan sulphate proteoglycans, the synthesis of which remains unaffected. Compared with the product from monolayer cultures, small proteoglycan II from collagen gels contained a longer polysaccharide chain which is characterized by a larger proportion of disulphated and a smaller proportion of monosulphated glucuronic acid-containing disaccharides. The half-life varied between 60 and 110 h. It is suggested that the compositional differences between the proteoglycan from monolayer cultures and from cells in a collagen lattice are related to the slower intracellular trafficking of the proteoglycan under the latter culture conditions.

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts.

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.

Mechanical confinement inhibits collagen synthesis in gel-cultured fibroblasts.

Collagen synthesis in fibroblasts cultivated in collagen lattices is known to be repressed compared to synthesis in monolayer cultures on plastic. Inhibition of synthesis is supposed to be due to interactions between the plasma membrane and adjacent collagen fibrils. To evaluate how collagen synthesis is regulated in gel-cultured cells we cultivated fibroblasts within gels of polymerized alginate in which preexisting extracellular matrix components, e.g., type I collagen fibrils, were lacking. When alginate gels were examined at the ultrastructural level, normal collagen fibrils were not observed. However, broad sheets of microfibrillar material and so-called zebra bodies were found. The amount of collagen synthesized by fibroblasts in calcium alginate gels remained constant during the entire culture time and was about 70% of that produced in monolayer-cultured cells. This value corresponded to levels found in fully retracted collagen lattices on day 7 of culture. Our data suggest that interactions between the plasma membrane and adjacent collagen fibrils are not necessary for the inhibition of collagen synthesis. Thus, we present data that mechanical confinement is capable of inhibiting collagen synthesis in fibroblasts.

Binding and uptake of Col 1(I), a peptide capable of inhibiting collagen synthesis in fibroblasts.

Col 1(I), a collagenase-resistant segment of the amino-terminal propeptide of pro alpha 1(I) chains, is known to inhibit collagen synthesis in cultured skin fibroblasts and also in a cell-free protein synthesizing system by reducing the translation of procollagen mRNA. These findings prompted us to explore the fate of exogenous Col 1(I) in the cellular processing of human skin fibroblasts using colloidal gold labeled protein (Col 1(I)-Au). Distribution of Col 1(I)-Au on the cell surface was studied by the platinum-carbon replication technique. Three different types of binding pattern could be observed: 1) Binding sites in the form of a fibrillar network, 2) those in the form of clusters, and 3) solitary bound gold conjugates. The latter two cases were determined to be specific. The intracellular routing of Col 1(I)-Au was studied by thin sections. Specifically bound gold conjugates were found in coated pits and after the initiation of the internalization process in coated vesicles and endosomes. Acid phosphatase cytochemistry revealed that only a small amount of Col 1(I)-Au is delivered to lysosomes. The bulk of gold conjugates is present even after prolonged incubation at 37 degrees C in acid phosphatase-negative compartments of the cell. Our data suggest a mechanism in which Col 1(I) initially is bound to the cell surface and subsequently internalized via the coated pit-coated vesicle pathway.