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In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-?B (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The mRNA levels of both RANKL (p < 0.01) and RANK (p < 0.05) were high in peri-implant tissue and RANKL+ and RANK+ cells were found in such tissue. Double labelling also disclosed soluble RANKL bound to RANK+ cells. We were unable to stimulate fibroblasts to express RANKL in vitro, but monocyte activation with LPS gave a fivefold increase in RANK mRNA levels. In contrast to RANKL and RANK expression in peri-implant tissue, expression of OPG was restricted to vascular endothelium. Endothelial cell OPG mRNA levels were regulated by TNF-a and VEGF, but not by hypoxia. It is concluded that activated cells in the interface tissue overproduce both RANKL and RANK and they can interact without interference by OPG.

AbstractTo clarify the mechanisms involved in amyloid formation in Finnish-type familial amyloidosis (FAF), we have tested the in vitro fibrillogenicity of synthetic wild-type and mutated gelsolin peptide analogs and studied the fragmentation patterns of gelsolin in the circulation of FAF patients with the Asn-187 or Tyr-187 gelsolin mutation.Fibril formation of synthetic peptides having sequence homology with wild-type or mutant gelsolins was monitored by Congo-red staining and polarization microscopy, negative staining electron microscopy and quantitative thioflavine-T fluorometry. Immmunoblotting with anti-gelsolin and amyloid-specific antibodies and sequence analyses were used to study the fragmentation pattern of gelsolin.Ultrastructurally amyloid-like fibrils were formed from mutant Asn-187 and Tyr-187 gelsolin peptides. Fluorometric analysis revealed highly accelerated fibril formation from the mutant peptides as compared with the corresponding wild-type peptides. Addition of mercaptoethanol alone or in combination with dithiotreitol tended to enhance fibril formation of the 9-mer and 11-mer Asn peptides. Blocking of the C-terminal carboxyl of the mutant Asn-187 gelsolin182-192peptide by amidation increased amyloidogenicity. The Tyr-187 gelsolin mutation, corresponding to the naturally occurring mutation in the Danish subtype of FAF, required acidic conditions to form fibrils meeting the criteria of amyloid. In FAF patients, in addition to the full-sized gelsolin, a series of lower-molecular mass C-terminal fragments of gelsolin (70,000 45,000 Da) was found in the circulation. In homozygous FAF(Asn-187) the 65-kDa fragment containing the amyloid forming region and the 55-kDa fragment, devoid of that region, was the major gelsolin species in the plasma.The results indicate that the 65-kDa gelsolin fragment derived by α-gelsolinase cleavage at the mutation-induced novel proteolysis site Argl 72-Ala 173 represents the putative circulating precursor protein of tissue amyloid in FAF and that the Asp 187Asn/Tyr substitution in gelsolin creates a conformation that is highly fibrillogenic.

Objective.to study the circulating levels of interleukin-18 (IL-18), a proinflammatory cytokine implicated in the T helper 1 response, inpatients with rheumatoid arthrtitis (RA) with or without amyloidosis.Methods.Plasma IL-18 levels were studied by enzyme-linked immusorbent assay in 55 RA patients with reactive amyloidosis and in 55 RA patients without amyloidosis matched with respect to age, sex, seropositivity, disease duration and inflammatory activity, as well as in 55 healthy control subjects.Results.Plasma IL-18 levels were significantly elevated in RA patients as compared with control subjects. Those RA patients who had amyloid had significantly higher circulating level of 1L-18 than those without amyloid (418.1 ± 32.1 ng/l versus 317.0 ± 21.3 ng/l, P<0.02). This difference was not due to differences in inflammatory activity, nor was it related to renal function.Conclusion.RA is associated with increased levels of plasma IL-18, the levels being significantly higher in patients with amyloid than in those without amyloid. The increased level in the amyloidosis patients may reflect the interaction of amyloid with cellular meatbolic pathways or, possibly, suggest a direct role of IL-18 in amyloidogenesis.

BackgroundFamilial amyloidosis of the Finnish type (FAF, Finnish hereditary amyloidosis) is caused by a 654G-A mutation in the gelsolin gene on chromosome 9 resulting in the expression of mutant Asn-187 gelsolin which is abnormally proteolytically processed generating amyloidogenic fragments that polymerise into amyloid fibrils. We have recently shown that in a Danish and a Czech family with a clinical syndrome similar to FAF, including corneal lattice dystrophy, cranial neuropathy and skin changes, the disease is caused by another mutation at the same position, namely 654G-T predicting a Tyr-for-Asp substitution at 187 in secreted gelsolin.AimTo undertake a closer examination of the Danish subtype of FAF and report immunohistochemical and biochemical findings.ResultsImmunostaining of plasma gelsolin isolated from heterozygous FAF of the Danish subtype revealed a pattern similar to that found in FAF-Asn 187. The > 60 kDa gelsolin species contain an epitope characteristic of the amyloid forming region as revealed by an amyloid specific antibody, whereas the ∼50 kDa fragments are devoid of it. Compared with the wild-type gelsolin peptide (Asp-187), the corresponding mutant peptide (Tyr-187) showed dramatically increased fibrillogenicity as revealed by quantitative thioflavine-T based fluorimetry; ultrastructurally, amyloid-like fibrils were formed by the mutant peptide. Immunohistochemistry showed that antibodies directed against residues 231-242 of secreted gelsolin, representing the carboxy terminus of the sequence forming the amyloid protein (residues 173-243) laid down in the tissues in a fibrillar form in FAF, specifically labelled the amyloid deposited in rectum and skin in the Danish (654G-T) subtype.ConclusionsThe 654G-T mutation in the gelsolin gene gives rise to an amyloid disease clinically and pathogenetically similar to that caused by the 654G-A mutation.

A comparison between viscosities in the systems sodium octanoate — water —n-decanol/n-pentanol is made. It is found that the isoviscosities behave quite differently. In the case ofn-decanol as co-surfactant the isoviscosities rapidly increases as the phase border towards the mesophaseF is reached. In the pentanol system we register no large increase in the viscosity for higher micellar contents. The effect of an addition withn-octane to the two systems is also quite different. The decrease in viscosity, as the content ofn-octane is increased, is much more pronounced for the long-chain alcohol system. These data support earlier findings that then-pentanol system should exhibit a much lower content of micellization.

Mutations in the presenilin 1 gene on chromosome 14 and in the related gene on chromosome 1 have been identified in individuals with early-onset familial Alzheimer's disease. The functions of the presenilin gene products as well as the relation of the presenilin mutations to amyloidogenesis are unclear. Here we show that peptides homologous to two disease-associated mutant forms of presenilin 1 and 2, respectively, show highly increased amyloid fibril formation as compared with the wild-type peptide homologues. The 410 Cys →Tyr (S-182) 14-residue peptide and the 141 Asn→Ile (E5-1) 15-residue peptide spontaneously assemble to fibrils of 7 to 9 nm width with strong Congophilic characteristics. Thioflavine-T fluorometry reveals a 7- to 18-fold higher rate of amyloid fibril formation of the mutant peptides as compared with the corresponding wild-type homologues. The results provide new insights into the mechanisms by which presenilin mutations lead to Alzheimer-type neuropathology: missense mutations in the presenilin genes may create products that are intrinsically highly amyloidogenic and directly involved in pathogenesis.

&lt;it>Objective&lt;/it>. To investigate the involvement of a disintegrin and the metalloproteinase ADAM9 (meltrin-γ) in the formation of multinuclear giant cells and osteoclasts in aseptic loosening of hip replacement implants. &lt;it>Methods&lt;/it>. We used &lt;it>in situ&lt;/it> hybridization, immunohistochemical staining and western blotting of interface membrane surrounding loosened hip implants, macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) costimulation and polymethyl methacrylate (PMMA) particle stimulation of human monocytes followed by immunofluorescence staining and flow cytometric analysis. &lt;it>Results&lt;/it>. Morphometric analysis revealed that the ADAM9&plus; area in the revision total hip replacement (THR) interface was larger than in primary THR samples (37.6±5.1 &lt;it>vs&lt;/it> 5.2±0.8&percnt;, &lt;it>P&lt;/it>&equals;0.002). Double immunofluorescence staining showed that CD68&plus; interface tissue macrophages and multinuclear giant cells were ADAM9&plus;. ADAM9 mRNA containing mononuclear and multinuclear cells was often seen in a close spatial relationship with other ADAM9&plus; cells. Western blotting disclosed a 50 kDa ADAM9 band in tissue extracts. Upon M-CSF and RANKL costimulation of human monocytes, the ADAM9 staining pattern changed over time and ADAM9&plus; cells formed bi- and multinuclear cells. Flow cytometry disclosed that cells of the monocyte&sol;macrophage lineage changed from ADAM9-negative cells into strongly positive cells during a 3-day culture. &lt;it>Conclusion.&lt;/it> ADAM9 is expressed in interface tissues around aseptically loosened THR implants. ADAM9 may play a role as a fusion molecule in the formation of multinuclear giant cells and osteoclasts from mononuclear precursors in diseases characterized by bone tissue destruction.