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The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β 2 -microglobulin (D76N β 2 -m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β 2 -m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β 2 -m expressing worms. We also demonstrated the specificity of the β 2 -m variant in determining the pathological phenotype by rescuing the wild type phenotype when β 2 -m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.

The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: β2-microglobulin and Aβ peptide3-42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm's life cycle (LC50 survival, 0.9 µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer's disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a "fragile population", where an underlying pathologic state may amplify their toxicity.

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β(2)-microglobulin (β(2)-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β(2)-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β(2)-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β(2)-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β(2)-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β(2)-m.

Background: We have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI. Therefore, based on the peculiar differences of the two tryptophans, we have analysed the evolution of β2-microglobulin with respect to these residues., Results: Having defined the β2-microglobulin protein family, we performed multiple sequence alignments and analysed the residue conservation in homologous proteins to generate a phylogenetic tree. Our results indicate that Trp60 is highly conserved, whereas some species have a Leu in position 95; the replacement of Trp95 with Leu destabilizes β2-microglobulin by 1 kcal/mol and accelerates the kinetics of unfolding. Both thermodynamic and kinetic data fit with the crystallographic structure of the Trp95Leu variant, which shows how the hydrophobic cavity of the wild-type protein is completely occupied by Trp95, but is only half filled by Leu95., Conclusions: We have established that the functional Trp60 has been present within the sequence of β2-microglobulin since the evolutionary appearance of proteins responsible for acquired immunity, whereas the structural Trp95 was selected and stabilized, most likely, for its capacity to fully occupy an internal cavity of the protein thereby creating a better stabilization of its folded state.

The protein chemistry laboratory of the Pavia University Hospital is specialized in the study of monoclonal components in body fluids. It is closely connected with the research laboratory devoted to the structural and functional study of pathogenic proteins and with the Clinical Chemistry Central Laboratory. The analyses are performed on specific medical request. The analytical approach is mainly based on analysis of patient's serum and urine by high-resolution agarose gel electrophoresis and immunofixation with the possible addition of the quantification of specific proteins. The interpretation of the protein pattern and the final reporting result from the integration of the laboratory data with the clinical information. This labor-intensive approach requires skills in the performance of protein analysis and in the interpretation/referral phase, as well as close communication with the attending physician.

We identified a novel missense mutation in the apolipoprotein A-I gene, T2069C Leu(174) --> Ser, in a patient affected by familial systemic nonneuropathic amyloidosis. The amyloid deposits mostly affected the heart of the proband, who underwent transplantation for end-stage congestive heart failure. Amyloid fibrils of myocardial and periumbilical fat samples immunoreacted exclusively with anti-ApoA-I antibodies. Amyloid fibrils extracted from the heart were constituted, according to amino acid sequencing and mass spectrometry analysis, by an amino-terminal polypeptide ending at Val(93) of apolipoprotein A-I (apoA-I); no other significant fragments were detected. The mutation segregates with the disease; it was demonstrated in the proband and in an affected uncle and excluded in three healthy siblings. The plasma levels of high-density lipoprotein and apoA-I were significantly lower in the patient than in unaffected individuals. This represents the first case of familial apoA-I amyloidosis in which the mutation is outside the polypeptide fragment deposited as fibrils. Visualization of the mutation in the three-dimensional structure of lipid-free apoA-I, composed of four identical polypeptide chains, indicates that position 174 of one chain is located near position 93 of an adjacent chain and suggests that the amino acid replacement in position 174 is permissive for a proteolytic split at the C-terminal of Val(93).

Circulating clonally related earlier cells are present in multiple myeloma (MM) and may be involved in the dissemination of this disease, its recurrence, and chemoresistance. The nature, stage of differentiation, and size of this cell population remain uncertain. Unlike other B-cell markers, CD22 membrane expression is limited to the late differentiation stages comprised between mature B cells (CD22+) and plasma cells (PC; CD22-) and may thus be useful in delimiting the maturational state of circulating early myeloma cells. Peripheral blood clone-related cells were detected by anti-idiotypic (Id) monoclonal antibodies and found to express CD22 (92% to 95%), monotypic light and heavy chain (100%), and CD38 (45%), whereas bone marrow PC were CD22-negative. CD22 expression was also documented on functional myeloma PC precursors, defined as peripheral blood cells capable of in vitro cytokine-driven monotypic PC differentiation, because up to approximately 70% inhibition of this process was obtained in 10 myeloma patients through the use of biospecific antibodies (BsAb) that deliver the plant toxin saporin to CD22+ cells. As further evidence of CD22 on circulating abnormal cells, it was found that in the only patient analyzed for DNA content, a portion of the peripheral blood CD22+ cells killed were hyperdiploid. Collectively, these findings indicate that most peripheral blood myeloma PC precursors are mature or later B cells presenting membrane CD22. The pattern of CD22 expression suggests the existence in MM of a differentiation process analogous to the normal antigenic response, in which CD22+ B cells migrate to the bone marrow and lose CD22 with PC differentiation. In addition, the sensitivity of myeloma PC precursors to the cytotoxic effects of the anti-CD22 BsAb and the possibility of interfering with their differentiation have potential therapeutic relevance.

Immunochemical study of the cryoglobulins has produced a classification of three main types which present clinical correlates. With the current standard immunofixation technique the vast majority of cryoglobulins are presently classified as type II. High-resolution immunoelectrophoretic techniques (immunoblotting and two-dimensional polyacrylamide gel electrophoresis) have helped to identify new sub-types whose clinical relevance is as yet undetermined. Nevertheless, these sensitive techniques could contribute to elucidate the various stages in the natural history of HCV-related cryoglobulins, from possible earlier HCV-antigen-driven polyclonal B cell proliferation, to autonomous monoclonal benign, to malignant B cell proliferation.

Background: Routine electrophoretic analysis fails to detect a monoclonal component (MC) in a considerable portion of AL amyloidosis patients. We investigated whether the combination of immunofixation (IF) on agarose gel electrophoresis and bone marrow plasma cell (BMPC) light chain kappa/lambda ratio analysis could contribute to diagnosis in these cases. The possible use of the BMPC kappa/lambda ratio in monitoring the clone was also investigated., Methods: We performed BMPC kappa/lambda ratio analysis and IF of serum and urine in 16 selected patients with no detectable MC at routine analysis, despite clinical features suggestive of primary amyloidosis. An anti-idiotypic monoclonal antibody specific for the amyloidogenic immunoglobulin and the BMPC kappa/lambda ratio were used to monitor the clone in a patient who underwent autologous peripheral blood stem cell transplantation., Results: Abnormal kappa/lambda ratios were found in 14 (sensitivity 87.5%), and a MC in 12 (sensitivity 75%). Combination of the two analyses confirmed diagnosis in all cases. In one patient changes in the size of the clone, monitored on serial bone marrow aspirates by an anti-idiotypic antibody, paralleled variations of the kappa/lambda ratio., Conclusions: This study demonstrates that the combined use of IF and the BMPC kappa/lambda ratio is extremely powerful in AL amyloidosis. In addition, the BMPC kappa/lambda ratio should be considered for monitoring the amyloidogenic clone when serum or urine MC is not quantifiable.

Background: AL amyloidosis is characterized by systemic tissue deposition of monoclonal Ig light chains synthesized by a bone marrow plasma cell (PC) clone whose biologic characteristics remain undetermined., Experimental Design: Anti-idiotypic (anti-Id) monoclonal antibodies (MoAbs) were used as specific probes to identify and study amyloidogenic cells in two patients by means of immunofluorescence methods. These MoAbs recognized populations of bone marrow pre-PC, PC, and peripheral blood lymphocytes. To test whether the circulating Id+ lymphocytes were capable of PC differentiation, peripheral blood lymphocytes were incubated with the differentiation-inducing agents, interleukin-3 and interleukin-6 in liquid culture. Preincubation with the anti-Id MoAb and complement was used to inhibit formation of Id+PC in vitro., Results: The anti-Id MoAb identified three types of cells in the bone marrow with cytoplasmic Ig having the same isotype as the monoclonal component: a) lymphoid cells, that were slightly larger than common peripheral blood lymphocytes (47% CD45RA+, 28% CD45R0+, 97% CD38-, 100% CD10-, 100% mu-chain-); b) lymphoplasmacytoid cells with more abundant cytoplasm and Id+ Ig (CD45RA-, CD45RO-, CD10-, 53% CD38+); 3) mature PC that were very similar to normal PC in morphology and antigenic profile (CD38+, PCA1+, CD56-). A different picture was seen when anti-Id MoAb were used to detect peripheral blood Id+ elements: analysis revealed a population of mature resting surface Ig+ B lymphocytes. Circulating Id+ lymphocytes differentiated in vitro to PC and lymphoplasmacytoid cells that were very similar to those present in the bone marrow. A significant reduction in the number of Id+ PC was obtained after incubation with the anti-Id MoAb and complement., Conclusions: This study shows that the amyloidogenic cell clone is constituted by at least the following cell populations: a fraction of bone marrow cells (lymphoid, lymphoplasmacytoid cells and PC) and a subset of peripheral blood post-switched B lymphocytes. The results suggest a relationship among these cells, indicating that circulating Id+ lymphocytes may be the possible precursors of the more differentiated bone marrow population.

A monoclonal anti-idiotyped antibody (IgG1k MAb 3B11D4) has been raised against the lambda-chain dimers isolated from the urine of a patient (DEP) with AL amyloidosis. This antibody binds a conformational idiotope present on the monoclonal DEP IgA, but does not recognize the reduced and alkylated lambda-chain monomers, nor the 15- to 17-kDa light chain fragments obtained from the amyloid fibrils, which have the same N-terminal sequence as the urinary light chains. The nonreactivity of this MAb with amyloid fibrils was confirmed by immunohistochemical examination of cryostatic sections of an amyloidoma surgically removed from the patient's subcutaneous tissue. Our data demonstrate that the deletion of about 70 amino acid residues of the C-terminus of the lambda chain prevents the formation of the self-limiting dimer and may facilitate the deposition of fragments into amyloid fibrils. With regard to the amyloidogenic clone, MAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning and cytotoxicity experiments demonstrate that this antibody has the capability of selectively eliminating the idiotype-positive cells from peripheral blood. Antibodies with these properties could find application in a new therapeutic strategy which provides high-dose chemotherapy, total body irradiation, and rescue with circulating stem cells. These antibodies could be used in two distinct phases: first, in the purging of the stem cells to be infused from the amyloidogenic clone and, secondly, in an attempt to eliminate residual disease by intravenous infusion.

In a patient (BAR) affected by chronic active hepatitis we isolated a cryoglobulin constituted of polyclonal IgM (k and lambda) and a monoclonal IgG3 lambda. The IgM represented the antibody of the cryoglobulin complex. This type of cryoglobulin, where the monoclonal component is the antigen and not the antibody, cannot be correctly classified using the nomenclature of Brouet et al. (1) currently in use. In this patient a two-year follow-up excluded any clinical signs of cryoglobulin toxicity. The immunochemical and clinical characterization of other cryoglobulins similar to BAR could establish a new homogeneous group.

The immunosubtraction technique was used to identify the proteins in electrophoretically separated plasma. The technique consists of forcing the plasma through a layer of monospecific antibody purified by electrophoresis; the specific protein is thereby precipitated and is consequently absent from the subsequent electrophoretogram. This technique was successfully applied to the following proteins (in order of distance from the anode): prealbumin, albumin, alpha-lipoproteins, alpha 1-antitrypsin, Gc-globulins, alpha 2-macroglobulin, haptoglobin, fibronectin, transferrin, beta-lipoproteins, C3, fibrinogen. The method may be applied to the study of protein polymorphism.