Amyloid Fibrils In Patients Research Articles

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains

BackgroundIn a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity.ResultsWe introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins.ConclusionsWe show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1–83) of apoA-I containing this mutation deposit as amyloid fibrils in patients’ tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.

Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain.

Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer.

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (VL) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of VL domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the VL domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol(-1), which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in VL domains of the κ family and not in VLλ or in VH domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.

Resident Macrophages Mediate Islet Amyloid Polypeptide–Induced Islet IL-1β Production and β-Cell Dysfunction

Islet amyloid polypeptide (IAPP) aggregates to form amyloid fibrils in patients with type 2 diabetes and acts as a potent stimulus for interleukin (IL)-1β secretion by bone marrow-derived macrophages. We sought to determine the contribution of resident islet macrophages to IAPP-induced inflammation and β-cell dysfunction. In cultured islets, macrophages (F4/80(+)CD11b(+)CD11c(+) cells) were required for IAPP-induced mRNA expression of the proinflammatory cytokines IL-1β, tumor necrosis factor-α, and IL-6 and the anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist. Moreover, IAPP-induced IL-1β synthesis and caspase-1 activation were detected in macrophages but not other islet cell types. Transgenic mice with β-cell human IAPP (hIAPP) expression had impaired glucose tolerance, elevated islet Il1b mRNA, and decreased Il10 and Il1rn expression following high-fat feeding. Islet macrophages were the major source of these transcripts and expressed increased cell surface Ly6C and CD11c in hIAPP transgenic mice. Clodronate liposome-mediated depletion of islet macrophages improved glucose tolerance and blocked proinflammatory gene expression in hIAPP-expressing mice, despite increasing the amount of islet amyloid. These data provide the first evidence that IAPP aggregates skew resident islet macrophages toward a proinflammatory phenotype and suggest a mechanism by which anti-inflammatory therapies may protect β-cells from IAPP-induced islet dysfunction.

Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly.

The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition--incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein--modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.

Molecular dynamics simulations to investigate the relationship between the structural stability and amyloidogenesis of the wild-type and N-terminal hexapeptide deletion ΔN6 β2-microglobulin

β2-Microglobulin (β2m) forms amyloid fibrils in patients undergoing long-term haemodialysis, leading to dialysis-related amyloidosis. Proteolysis of the N-terminal region of β2m results in a truncation of the six N-terminal residues (ΔN6 β2m) in ∼30% of the β2m molecules extracted from ex vivo fibrils. The ΔN6 β2m has been shown to exhibit a higher tendency for self-association comparing to the wild-type (wt) β2m, particularly at neutral pH. In order to gain atomic insights into the early stages of amyloid formation of the wt and ΔN6 β2m, various molecular dynamics simulations were conducted to investigate the stability and dynamics of these two molecules at various temperatures and neutral pH in this study. Our results, in agreement with previous experimental results, indicate that the structural stability of the ΔN6 β2m is lower than that of the wt β2m. It can be attributed to fact that the removal of the N-terminal six residues results in the loss of the salt–bridge interaction between residues R3 and D59, leading to the increased solvent exposure of the K3 peptide. It further allows water molecules to destabilise the interior region of the K3 peptide, leading to the elongation between the B- and E-strands. It may further accelerate the conformational changes of the ΔN6 β2m, leading to the formation of amyloid fibrils more readily at neutral pH. Our results also suggest that the K3 peptide may be a potential initiation site of amyloid formation for the ΔN6 β2m due to its increased solvent exposure. We further suggest that fibril morphology of the ΔN6 β2m formed at neutral pH is similar to that of the wt β2m formed at low pH (1.5–3) since they adopt the similar conformation with the elongation between B- and E-strands for their partially unfolded amyloidogenic intermediates.

Molecular Dynamics Simulations to Gain Insights into the Stability and Morphologies of K3 Oligomers from β2-microglobulin

β2-Microglobulin (β2-m) forms amyloid fibrils in patients undergoing long-term hemodialysis. K3 peptide, a Ser20-Lys41 fragment of β2-m, has been known to form fibrils over a wide range of pH and solvent conditions. Recent solid-state NMR has revealed that K3 oligomer adopts a parallel U-shaped β-strand-turn-β-strand motif. In order to investigate the stability and morphologies of K3 oligomers with different sizes (dimer, trimer, and tetrameri and organizations (single and double layers), several all-atom molecular dynamics simulations were conducted at 310 K and pH 2 in water and 2,2,2-trifluoroethanol (TFE). For single-layered organizations, our results show that TFE destabilizes the stacking of K3 peptides due to the fact that TFE weakens the intermolecular hydrophobic interactions of K3 oligomers. In addition, we also identified that the loop region is stabilized by the hydrophobic cluster involving resides Y7, Fll, and I16. Our results further suggest that K3 tetramer is a potential minimal nucleus seed for the formation of K3 protofibrils. For dou-ble-layered organizations in water, our data demonstrate that K3 peptides can form various stable assemblies through different interfacial arrangements, such as NN, NC, and CC, by different driving forces. We further propose that the stacking of different interfaces between two facing β-sheets of K3 peptides could be related to different fibril morphologies, which is in good agreement with the previous experimental results, showing that K3 protofibrils associated to formed mature fibrils with a wide range of diameters from 4 to 15 nm when they were transferred from 20% (v/v) TFE to aqueous solution.

Serum Immunoglobulin Free Light Chain in Primary Amyloidosis: Prognostic Value and Correlations with Clinical Features

Background: Immunoglobulin free light chains (FLC) form the substrate for synthesis of amyloid fibrils in patients with AL amyloidosis. Development of FLC assay (Freelite) has allowed us to better assess the clonal cell burden in patients with AL amyloid. The relationship of the light chain types and their levels in serum to the clinical feature at presentation and the eventual outcome has not been systematically studied.Methods: We identified 730 patients with biopsy proven AL amyloidosis, who were seen at Mayo Clinic between January 1980 and July 2006, who were seen within 90 days of their diagnosis and in whom FLC levels were performed or had stored serum available for analysis. Cardiac biomarkers (cTnT, NT ProBNP) were also performed on stored serum for some of the patients as part of previous studies. Clinical data and follow up status are prospectively collected into the Dysproteinemia database, which was used for the study.Results: The median age of the study population was 63 years (range; 32–90) and 463 patients (63%) were male. The median estimated survival for the entire group was 37 months from diagnosis and the median follow up for the 263 patients (37%) alive at the time of the analysis was 27.8 months (range; 1–177 months). The plasma cell clone was lambda in 528 patients (72%) and kappa in 202 (28%) patients. The median absolute difference between the FLCs was 19.6 mg/dL for the entire group (range; 0.01–2478); for the kappa patients was 29.4 mg/dL (range; 0.01–1359) and for the lambda patients was 18.2 mg/dL (range; 0.03–2478). Kappa AL was associated with more involvement of the GI tract and liver as indicated by higher alkaline phosphatase (1.8 vs. 1.3 fold; P=0.03), higher total bilirubin (0.95 vs. 0.78 mg/dL, P = 0.04) and lower serum carotene (119 vs. 155 ug/dL; P = 0.0002). On the contrary, renal involvement was more in the lambda AL with the 24 hour urinary albumin higher in the lambda AL (2.4 gm vs. 1.4 gm; P = 0.0002) and lower serum albumin (2.77 vs. 2.97 mg/dL; P = 0.001). While there was no difference in the overall survival (OS) between the kappa and lambda AL, the median OS for those without a heavy chain was significantly shorter (12.6 months vs. 29.9 months; P= 0.01) compared to those with a heavy chain identified. There was also a correlation between high FLC difference and degree of organ involvement, especially cardiac. Among the 202 kappa AL patients, the median survival for those with a high FLC difference ( > 29.4 mg/dL) was 13 months vs. 48.8 months for those with low difference (P = 0.001). Similarly among the 528 patients with lambda AL, those with high difference (> 18.2 mg/dL) had a median OS of 9.3 months compared to 33 months for those with low difference (P < 0.0001) (Figure). The FLC difference was independent of the cTnT, NT Pro BNP, uric acid and fold-increase in alkaline phosphatase, all of which were significant in the multivariable analysis.Conclusions: The type of FLC impacts the spectrum of organ involvement, with gastrointestinal involvement observed more frequently with kappa, and renal involvement with lambda FLC. The FLC burden correlates with the degree of organ involvement, and is a significant predictor of overall survival in AL amyloidosis. Lambda FLC exerts similar effect as kappa FLCs, but at lower levels confirming the increased “amyloidogenicity” of lambda FLC. Finally, lack of a chaperone heavy chain is associated with a poorer outcome. [Display omitted]

High-resolution Crystal Structure of 2-Microglobulin Formed at pH 7.0

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex class I, forms amyloid fibrils in patients undergoing long-term haemodialysis, causing dialysis-related amyloidosis. Based on a comparison of the X-ray structure obtained at pH 5.7 and that of beta2-m in the histocompatibility complex, it has been proposed that the continuous D-strand observed in the crystal structure at pH 5.7 increases the propensity of beta2-m to self-associate via edge-to-edge interactions, thus initiating the formation of fibrils. To obtain further insight into the mechanism by which amyloid fibrils form, we determined the crystal structure of beta2-m at pH 7.0 at a resolution of up to 1.13 A. The crystal structure at pH 7.0 was basically the same as that at pH 5.6, suggesting that the conversion of the beta-bulge in strand D into a contiguous beta-strand is not unique to the crystals formed under slightly acidic conditions. In other words, although the formation of beta2-m fibrils was enhanced under acidic conditions, it remains unknown if it is related to the increased propensity for the disappearance of the beta-bulge in strand D. We consider that the enhanced fibrillation is more directly coupled with the decreased stability leading to the increased propensity of exposing amyloidogenic regions.

Investigating the structural properties of amyloid‐like fibrils formed in vitro from β2‐microglobulin using limited proteolysis and electrospray ionisation mass spectrometry

The protein beta(2)-microglobulin (beta(2)m) aggregates to form classical amyloid fibrils in patients undergoing long-term haemodialysis. Amyloid-like fibrils with a cross-beta fold can also be formed from wild-type beta(2)m under acidic conditions in vitro. The morphology of such fibrils depends critically on the conditions used: incubation of beta(2)m in low ionic strength buffers at pH 2.5 results in the formation of long (microm), straight fibrils while, at pH 3.6, short (<500 nm) fibrils form. At higher ionic strengths (0.2-0.4 M) at pH 1.5-3.6, the fibrils have a distinct curved and nodular morphology. To determine the conformational properties of beta(2)m within in vitro fibrils of different morphologies, limited proteolysis of each fibril type using pepsin was performed and the resulting peptide fragments identified by tandem mass spectrometry. For comparison, the proteolytic degradation patterns of monomeric beta(2)m and seven synthetic peptides spanning the entire sequence of the intact protein were similarly analysed. The results show that fibrils with different morphologies result in distinct digestion patterns. While the curved, worm-like fibrils are relatively weakly protected from proteolysis, the long, straight fibrils formed at pH 2.5 at low ionic strength show only a single cut-site at Val9, demonstrating that substantial refolding of the initially acid-denatured and unprotected state of beta(2)m occurs during assembly. The data demonstrate that the organisation of the polypeptide chain in fibrils with different morphological features differs considerably, despite the fact that the fibrils possess a common cross-beta architecture.

Ultrasonication-induced Amyloid Fibril Formation of β2-Microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.

Amyloidosis and Lung Cancer

Clinical systemic amyloidosis has been rarely described in patients with lung cancer. A new case of such an association is described with a review of the literature and a discussion about the possible biological mechanisms responsible for the development of systemic amyloidosis in patients with lung cancer. Circulating precursors of amyloid fibrils in patients with lung cancer might be implicated more than expected in the biology of tumoral disease.

Analysis of transthyretin amyloid fibrils from vitreous samples in familial amyloidotic polyneuropathy (Val Met)

The aim of the present study was to analyze the forms of wild type and mutated monomeric transthyretin (Val3 et) in the amyloid fibrils of patients with familial amyloidotic polyneuropathy by electrospray ionization mass spectrometry (ESI-MS).The solubility of amyloid fibrils from the vitrectomized samples was examined to determine the appropriate solution for ESI-MS. ESI-MS analysis revealed that heterozy-gotic Val3 et amyloid fibrils contained 14.6 ± 7.5 % normal TTR. In all samples, 3 different types of variant ATTR could be identified: Full length ATTR, and-57, and-157 (or 156) Da from ATTR Val3 et were found. The two peaks showing-57, and-157 (or 156) Da from ATTR Val3 et corresponded to the-Gly, and-Gly-Pro sequences of ATTR Val3 et from the N-terminal.The results illustrate the heterogeneity of ATTR amyloid deposits and this method may be very useful for analyzing amyloid fibrils in ATTR related amyloidosis.

Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis.

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.

Monocyte/macrophage response to β2-microglobulin modified with advanced glycation end products

We recently found that acidic beta 2-microglobulin (beta 2m), a major isoform of beta 2m in amyloid fibrils of patients with dialysis-related amyloidosis (DRA), contained early Amadori products and advanced glycation end products (AGEs) formed nonenzymatically between sugar and protein. Further analysis revealed that acidic beta 2m induces monocyte chemotaxis and macrophage secretion of bone-resorbing cytokines, suggesting the involvement of acidic beta 2m in the pathogenesis of DRA. Acidic beta 2m, however, is a mixture of heterogeneous molecular adducts due to various types of modification. In the present study, we investigated the modification responsible for the biological activity of acidic beta 2m toward monocytes/macrophages. The presence of a fair amount of beta 2m species with deamidation was detected in acidic beta 2m isolated from urine of non-diabetic long-term hemodialysis patients, but deamidated beta 2m had no biological activity. In contrast, normal beta 2m acquired the activity upon incubation with glucose in vitro. Among the glycated beta 2m, the pigmented and fluorescent beta 2m that formed after a long incubation period, that is, AGE-modified beta 2m, exhibited biological activity, whereas beta 2m modified with Amadori products, major Maillard products in acidic beta 2m, had no such activity. These findings suggest that AGEs, although only a minor constituent of acidic beta 2m, are responsible for monocyte chemotaxis and macrophage secretion of cytokines, implicating the contribution of AGEs to bone and joint destruction in DRA.

Hemodialysis-Related Induction of β2-Microglobulin Synthesis and Release by Mononuclear Phagocytes

We have investigated beta 2-microglobulin (beta 2M) synthesis and release by blood leukocytes and isolated mononuclear phagocytes. Since beta 2M was discovered to form amyloid fibrils in patients undergoing long term, chronic hemodialysis and monomeric beta 2M levels in plasma of these patients are highly elevated, and since hemodialysis-related factors that increase beta 2M production are unknown, we evaluated beta 2M production by mononuclear phagocytes under a variety of conditions. We utilized a novel enzyme-lined immunoabsorbant assay to quantitate beta 2M release. Adherence of macrophages onto polystyrene or Cuprophan membranes does not induce beta 2M synthesis or release. In contrast, interaction of macrophages with lipopolysaccharide, gamma-interferon, tumor necrosis factor, or interleukin 1 induces synthesis or release. In contrast, interaction of macrophages with lipopolysaccharide, gamma-interferon, tumor necrosis factor, or interleukin 1 induces synthesis and release of beta 2M, indicating that beta 2M synthesis is increased during macrophage activation. Exposing leukocytes or macrophages to changes in osmotic or oncotic pressure induces a rapid release of beta 2M and interleukin 1 into the cellular medium. These results suggest that during hemodialysis, beta 2M production is more likely to result from endotoxin contamination, or osmotic and oncotic changes, rather than direct interaction of mononuclear phagocytes with cellulosic membranes.

Hemodialysis-Related Induction of Beta-2-Microglobulin and Interleukin-1 Synthesis and Release by Mononuclear Phagocytes

We have investigated beta 2-microglobulin (beta 2M) synthesis and release by blood leukocytes and isolated mononuclear phagocytes. Recent interest in beta 2M has developed since the discovery that this protein forms amyloid fibrils in patients undergoing long-term, chronic hemodialysis and that these patients have greatly elevated levels of monomeric beta 2M in their circulation. Since hemodialysis-related factors that increase beta 2M production are unknown, we evaluated beta 2M production by freshly prepared leukocytes and monocyte-derived macrophages under a variety of conditions. We utilized a novel enzyme-linked immunoabsorbant assay to quantitate beta 2M concentrations, and monitored interleukin-1 and beta 2M synthesis by immunoprecipitation. Incubation of leukocytes with Cuprophan or Hemophan does not increase beta 2M release. Adherence of macrophages onto polystyrene or Cuprophan membranes induces neither interleukin-1 nor beta 2M synthesis or release. In contrast, interaction of macrophages with lipopolysaccharide, gamma-interferon, tumor necrosis factor, or interleukin-1 induces synthesis and release of beta 2 M, indicating that beta 2 M synthesis is increased during macrophage activation. Exposing leukocytes or macrophages to changes in osmotic or oncotic pressure induces a rapid release of beta 2M and interleukin-1 into the cellular medium. These results suggest that during hemodialysis, beta 2M production is more likely to result from endotoxin contamination, or osmotic and oncotic changes, rather than direct interaction of mononuclear phagocytes with Cuprophan membranes.

Beta-2-microglobulin synthesis is increased during activation of human monocytes.

We have investigated the regulation of beta 2-microglobulin (beta 2-M) synthesis by monocytes. Recent interest in beta 2-M has developed since the discovery that this protein forms amyloid fibrils in patients undergoing long-term, chronic hemodialysis. The beta 2-M amyloidosis is linked to the greatly elevated levels of monomeric beta 2-M in their circulation. Since factors that govern beta 2-M release from plasma membranes are not known, we endeavored to evaluate beta 2-M release during monocyte activation. Utilizing a human monocyte-like cell line, U937, we studied the effect of bacterial toxin stimulation on levels of membrane, cell surface, and supernatant beta 2-M. We now present a novel method to purify beta 2-M, a solid-phase radioimmunoassay to measure soluble beta 2-M, and an ELISA to measure membrane beta 2-M. Using these methods we found that the levels of beta 2-M in the cell membrane or on the cell surface did not change during monocyte activation. However, activation did induce a significant increase in the concentration of beta 2-M in monocyte supernatants, indicating that beta 2-M synthesis by monocytes is increased during monocyte activation. These results suggest that monocyte activation by hemodialysis membranes may be a contributing factor to the observed increase in circulating beta 2-M levels.

Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of gamma-trace basic protein (cystatin C).

A gamma-trace variant protein is the major constituent of the amyloid fibrils in patients from Iceland with hereditary cerebral hemorrhage with amyloidosis. The protein consists of 110 residues and is similar to human urinary gamma-trace basic protein (or cystatin C) beginning at its 11th amino-terminal residue. It has an amino acid substitution (glutamine for leucine) at position 58 (position 68 in gamma-trace numbering), which is near the proposed active site of related proteins--namely, cysteine protease inhibitors and kininogens. It is postulated that a point mutation has occurred, leading to the production of an unusual protein that is abnormally degraded, bound, and/or precipitated. Alternatively, gamma-trace basic protein may be genetically polymorphic, and the variant described here may represent an as-yet-undiscovered isotype or an allelic form that is linked to, but not responsible for, the deposition disease. Our data on the structure of a gamma-trace variant protein suggests that its gene expresses a polyprotein precursor in which active peptides are flanked by basic amino acid residues that permit cleavage to liberate small internal peptides. It is likely that the nucleotide sequence coding for Arg-Xaa and Lys-Xaa repeated several times in the molecule may function as alternative splicing sites for mRNA processing.