Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis.
(49/1787)
Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution. (+info)
Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease.
(50/1787)
BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases. (+info)
Transforming growth factor-beta is involved in the pathogenesis of dialysis-related amyloidosis.
(51/1787)
BACKGROUND: Advanced glycation end product-modified beta2-microglobulin (AGE-beta2m) is an important component of dialysis-related amyloidosis (DRA). Its presence induces monocyte chemotaxis and the release of the proinflammatory cytokines through macrophage activation. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that also has chemotactic activity for monocytes at very low (0.1 to 10 pg/mL) concentrations and inhibits proinflammatory cytokine production of macrophages. In this study, we investigated the role of TGF-beta in the pathogenesis of DRA. METHODS: We performed an immunohistochemical study of DRA tissues (8 cases) to confirm the existence of TGF-betas and their receptors; we also performed a chemotaxis assay of human monocytes as well as enzyme-linked immunosorbent assay (ELISA) of TGF-beta1, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-1 receptor antagonist (IL-1Ra) in the supernatant of human monocyte-derived macrophage cell culture under varying conditions of incubation with TGF-beta1, AGE-beta2m, and TGF-beta1 antibody additions. RESULTS: There was positive staining for TGF-betas (types 1, 2, and 3) and their receptors (types I, II, and III) in infiltrated macrophages (CD68+), synovial lining cell, as well as vascular walls around amyloid deposition. AGE-beta2m also induced TGF-beta1 production by macrophages in a dose-dependent manner (410 +/- 80 pg/mL at 12.5 microg/mL, 621 +/- 62 pg/mL at 25 microg/mL, and 776 +/- 62 pg/mL at 50 microg/mL of AGE-beta2m). AGE-beta2m induced significant TNF-alpha and IL-1Ra production by macrophage. The addition of exogenous TGF-beta1 (0.1 to 10 ng/mL) decreased AGE-beta2m-induced TNF-alpha production and increased IL-1Ra production in a dose-dependent fashion. IL-1beta production was not effected by any experimental conditions. In chemotaxis assay, anti-TGF-beta1 antibody (0.1 to 10 microg/mL) attenuated AGE-beta2m-induced monocyte chemotaxis. CONCLUSIONS: These results provide the first evidence to our knowledge for the presence of TGF-beta in DRA tissue, as well as the stimulatory action of AGE-beta2m on tissue macrophages. In turn, TGF-beta suppresses the proinflammatory activation of macrophages, suggesting a dual role for TGF-beta in the inflammatory process of DRA. These observations may provide a pathophysiologic link between TGF-beta and DRA. (+info)
Bronchial amyloidosis successfully treated with low-dose long-term erythromycin therapy.
(52/1787)
A 69-year-old man was admitted for evaluation of an abnormal chest X-ray. A diagnosis of primary bronchial amyloidosis was made on the basis of the chest X-ray, CT scans and bronchial biopsy specimens. The patient was treated with low-dose long-term erythromycin therapy (600 mg/day). After four months of therapy, chest CT scans, bronchoscopic findings and bronchial biopsy specimens revealed significant improvement of inflammatory changes. Low-dose erythromycin therapy may be helpful in terms of its anti-inflammatory effects for patients with bronchial amyloidosis. (+info)
Molecular basis of a yeast prion species barrier.
(53/1787)
The yeast [PSI+] factor is inherited by a prion mechanism involving self-propagating Sup35p aggregates. We find that Sup35p prion function is conserved among distantly related yeasts. As with mammalian prions, a species barrier inhibits prion induction between Sup35p from different yeast species. This barrier is faithfully reproduced in vitro where, remarkably, ongoing polymerization of one Sup35p species does not affect conversion of another. Chimeric analysis identifies a short domain sufficient to allow foreign Sup35p to cross this barrier. These observations argue that the species barrier results from specificity in the growing aggregate, mediated by a well-defined epitope on the amyloid surface and, together with our identification of a novel yeast prion domain, show that multiple prion-based heritable states can propagate independently within one cell. (+info)
Genotype-phenotype correlation in a large group of Turkish patients with familial mediterranean fever: evidence for mutation-independent amyloidosis.
(54/1787)
OBJECTIVES: Differences in clinical manifestations of familial Mediterranean fever (FMF) between different ethnic groups have been documented. The FMF gene was recently cloned and four missense mutations (Met694Val, Met680Ile, Val726Ala, and Met694Ile) that account for a large percentage of the patients were identified. The results of initial mutation studies have led to the hypothesis that phenotypic variation of the disease may be attributable to the existence of some of these mutations. The purpose of this study was to evaluate whether this phenotypic variation is associated with the existence of particular mutations in Turkish FMF patients living in Turkey. METHODS: Four missense mutations and genotype-phenotype correlation were investigated in 167 Turkish FMF patients. The patients were grouped according to the presence of the Met694Val and the Met680Ile mutations, and 12 clinical parameters were compared between the groups. RESULTS: The presence of the Met694Val mutation was not found to be associated with a severe form of the disease or the development of amyloidosis. Arthritis frequency was found to be lower in the patients with homozygous Met680Ile mutation. CONCLUSIONS: None of the four missense mutations is associated with a severe disease or the development of amyloidosis in Turkish FMF patients living in Turkey. The influence of unknown environmental factors and/or the presence of other genetic changes are necessary to explain the phenotypic variation of the disease and the development of amyloidosis. (+info)
Clotting alterations in primary systemic amyloidosis.
(55/1787)
BACKGROUND AND OBJECTIVE: The bleeding manifestations frequently observed in patients with immunoglobulin light chain amyloidosis (AL) have been attributed to different pathogenetic factors: amyloid deposits in several organs and systems leading to failures of these latter, the affinity of amyloid for some clotting factors, and the presence of plasma components interfering with fibrin formation could all induce alterations of clotting tests. This investigation was aimed at defining the prevalence of clotting abnormalities and their clinical manifestations in patients with AL. DESIGN AND METHODS: Thirty-six consecutive patients with biopsy proven amyloidosis and documented monoclonal gammapathy were enrolled within one year. The following clotting tests were considered in the study: activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), Russell's viper venom time (RVTT), fibrinogen, factor X and alpha-2 antiplasmin. RESULTS: Hemorrhagic manifestations were mild to moderate in nine patients, but severe and untractable in one. The most frequent clotting anomaly was defective fibrinogen conversion to fibrin, as demonstrated by prolongation of both TT (85% of cases) and RT (90% of cases). Low levels of factor X activity were observed in about 1 out of 4 samples, while fibrinogen and alpha2 antiplasmin levels were distributed over a wide range of values. PT was prolonged in 8 and aPTT in 25 patients. The search for lupus anticoagulant was negative in samples showing a prolongation of aPTT and/or RVVT. INTERPRETATION AND CONCLUSIONS: The prolongation of TT and RT is not dependent on either the presence of a heparin-like substance in the plasma or on fibrinogen levels; furthermore, the prolongation of RVVT is not related to factor X level. The hypothesized presence in the plasma of an inhibitor of fibrin formation could also affect factor X activation by Russell viper venom. The prolongation of TT and RT represents a peculiar feature of amyloidosis. The variability in the behavior of the other clotting times and hemostatic factors studied is mirrored in the heterogeneity of the clinical features observed in this disease. (+info)
Myeloablative chemotherapy with stem cell rescue for the treatment of primary systemic amyloidosis: a status report.
(56/1787)
Stem cell transplantation has been incorporated in the treatment of primary systemic amyloidosis for 5 years. Results reported to date suggest that the response rates are substantially better than those for patients treated with low-dose traditional melphalan and prednisone chemotherapy. Unexpectedly high mortality rates have, however, been reported with stem cell transplantation, reaching 40% in some series. This unexpectedly high mortality appears to be related to multiorgan failure of tissues infiltrated with amyloid deposits. Deaths have been reported from gastrointestinal tract hemorrhage, gastrointestinal tract perforation, sudden cardiac death, and renal failure. The best patient for transplantation appears to have single organ involvement, an age <55 years, the absence of renal insufficiency, and no symptomatic cardiac dysfunction. Patients eligible to receive stem cell transplant represent a highly selected population, and before conclusions about the efficacy of transplantation are drawn, comparison with a matched control group is necessary. Amyloidosis should be considered an indication for stem cell transplantation in the context of a clinical trial so that results can be compiled and reported for an accurate assessment of response rate, survival, relapse rates and treatment-related toxicities. Bone Marrow Transplantation (2000) 25, 465-470. (+info)