Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. The importance and impact of the amyloid deposits are unknown. In this study we have purified a 5.5-kDa aortic medial amyloid component, by size-exclusion chromatography and RP-HPLC, from three individuals, and we have shown by amino acid sequence analysis that the amyloid is derived from an integral proteolytic fragment of lactadherin. Lactadherin is a 364-aa glycoprotein, previously known to be expressed by mammary epithelial cells as a cell surface protein and secreted as part of the milk fat globule membrane. The multidomain protein has a C-terminal domain showing homology to blood coagulation factors V and VIII. We found that the main constituent of aortic medial amyloid is a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By using in situ hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic peptides forms amyloid-like fibrils in vitro. Lactadherin was not previously known to be an amyloid precursor protein or to be expressed in aortic tissue. The structure of lactadherin may implicate an important regulatory function in the aorta.

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.

Previous studies have shown that the amyloid localized to the aortic intima may be a biochemical entity different from other forms of localized amyloid. The amyloid fibril protein in one patient studied consisted of an N-terminal fragment of apolipoprotein A-1 (apo A-1). Since this patient was later shown to carry a missense mutation in the apo A-1 gene, leading to a deletion at position 107 of the mature protein, the question remained whether wild-type apo A-1 is amyloidogenic. In autopsy specimens from the thoracic aorta from 69 individuals, intimal atherosclerotic plaque-related amyloid was present in 11 cases (16%) and amyloid outside plaques in 37 cases (54%). The immunoreactivity of amyloid localized to the aortic intima was evaluated with the aid of antisera against N-terminal segments of apo A-1. The amyloid in association with atherosclerotic plaques was positively labelled by immunohistochemistry. The amyloid fibril protein from one patient, previously shown not to carry any mutation in the apo A-1 gene, was purified and shown by amino acid sequence analysis to be of apo A-1 nature. The result shows that wild-type apo A-1 is amyloidogenic and gives rise to a common localized form of amyloid associated with atherosclerosis.

Small amyloid deposits commonly occur along the internal elastic lamina of the temporal artery. In temporal artery biopsies from 22 patients with histological signs of giant cell arteritis and 25 without, amyloid deposits were found in 14 and 21 biopsies, respectively. Two specific peptide antisera show that this amyloid is identical to the recently identified medin-amyloid in the ageing aorta. On immunoelectron microscopy, the amyloid appeared topographically closely related to the elastic material. Furthermore, fragmented elastic material was often immunolabelled for medin and found to be engulfed by giant cells. Medin is an internal fragment of the larger precursor lactadherin and is presumably formed by specific enzymatic cleavage events. In situ hybridization showed that lactadherin is expressed locally by smooth muscle cells of the temporal artery. Given the potential role of lactadherin as a mediator for the adhesion of cells, including macrophages, to other cells or surfaces, lactadherin or its fragment medin may be important in the inflammatory process in giant cell arteritis.

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SUMMARY T‐helper cells type 1 (Th1) and type 2 (Th2) play an important role in the pathogenesis of autoimmune diseases. In many Th1‐dependent autoimmune models, treatment with recombinant interleukin‐12 (rIL‐12) accelerates the autoimmune response. Mercury‐induced autoimmunity (HgIA) in mice is an H‐2 regulated condition with antinucleolar antibodies targeting fibrillarin (ANoA), systemic immune‐complex (IC) deposits and transient polyclonal B‐cell activation (PBA). HgIA has many characteristics of a Th2 type of reaction, including a strong increase of IgE, but disease induction is critically dependent on the Th1 cytokine IFN‐γ. The aim of this study was to investigate if a strong deviation of the immune response in HgIA towards Th1 would aggravate HgIA. Injections of both rIL‐12 and anti‐IL‐4 monoclonal antibody (α‐IL‐4) reduced the HgCl2‐(Hg‐)induced concentration of the Th2‐dependent serum IgE and IgG1, but increased the Th1‐dependent serum IgG2a. The IgG‐ANoA developed earlier and attained a higher titre after combined treatment, and the ANoA titre of the IgG1 isotype decreased while the ANoA titre of the Th1‐associated IgG2a, IgG2b and IgG3‐ANoA isotypes increased. Treatment with rIL‐12 alone increased the Hg‐induced IgG2a and IgG3 ANoA titres, the PBA, and the IC deposits in renal and splenic vessel walls, while treatment with α‐IL‐4 + Hg inhibited renal but not splenic vessel wall IC deposits. We conclude that manipulating the cytokine status, by altering the Th1/Th2 balance, will influence autoimmune disease manifestations. This might be an important way of modulating human autoimmune diseases.

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Cytokines play an important and complex role in the pathogenesis of systemic autoimmune diseases. In susceptible H‐2s mice, inorganic mercury (Hg) induces lymphoproliferation, antinucleolar antibodies against the 34‐kDa‐protein fibrillarin, and systemic immune‐complex (IC) deposits. Here, we report extensive analysis of cytokine mRNA levels in susceptible A.SW (H‐2s) and resistant A.TL (H‐2tl) mice under unstimulated conditions and during oral treatment with Hg and/or silver nitrate (Ag). Cytokine mRNA expression in lymphoid tissues was assessed using the ribonuclease protection assay and phosphorimaging. Baseline expression of IL‐2 and IFN‐γ mRNA was higher in A.SW than in A.TL mice. In A.SW mice, Hg treatment caused early up‐regulation of IL‐2 and IFN‐γ levels, followed by substantial expression of IL‐4 mRNA, which was significant compared to control A.SW and Hg‐treated A.TL mice. Hg‐exposed A.TL mice exhibited unchanged IFN‐γ, reduced IL‐2 and greatly increased IL‐10 mRNA expression. Ag‐treated A.SW mice, which develop antifibrillarin antibodies (AFA) but exhibit minimal immune activation and no IC deposits, showed an early increase in IL‐2 and IFN‐γ mRNA, but only a small and delayed rise in IL‐4 mRNA. In conclusion, H‐2‐linked resistance to Hg‐induced AFA is characterized by low constitutive expression of IL‐2 and IFN‐γ mRNA, which is not increased by Hg, and a marked increase in IL‐10 expression. Conversely, the key features of H‐2‐linked susceptibility to Hg‐ and Ag‐induced AFA are up‐regulation of IL‐2, IFN‐γ and IL‐4 mRNA expression, and down‐regulation of IL‐10 expression.