Irene Zorzoli

Light and electron microscopy immunohistochemical characterization of amyloid deposits

The present study reports our optimized fixation and processing methods for the light and electron microscopic immunohistochemical characterization of tissue amyloid. The study involved a series of 25 abdominal fat aspirates and of 11 tissue biopsy samples from different organs from 36 patients. We tested a short fixation and processing method and a series of enzymatic, acidic and physical (microwave oven) pretreatments on paraffin slides for light microscopy, Congo red stain and immunohistochemistry. Microwave antigen retrieval provided the highest enhancement for both Congo red stain and immunohistochemical reactions with all tested antibodies; formic acid also gave good results, but tissue morphology may have been altered. Immunoelectron microscopy provided evidence of fibril accumulation and immunoreactivity in both mixed (double immunoreactivity) and non-mixed forms, along with information on the relative amount of immunoreaction for the two components in mixed amyloid deposits; the technique also yi...

The Pavia approach to clinical protein analysis

Abstract 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 patients 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.

C. elegans Expressing Human β2-Microglobulin: A Novel Model for Studying the Relationship between the Molecular Assembly and the Toxic Phenotype

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 β2-microglobulin (β2-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 β2-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β2-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β2-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 β2-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 β2-m.

The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure.

BackgroundWe 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.ResultsHaving 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.ConclusionsWe 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.

Clonality and specificity of cryoglobulins associated with HCV: pathophysiological implications

BACKGROUND/AIMS Hepatitis C virus (HCV) infection plays a central role in the pathogenesis of mixed cryoglobulinemia through molecular mechanisms which remain to be elucidated. The aim of this study was to investigate the role of antibody responses to HCV in the pathogenesis of cryoglobulinemia through characterization of the anti-HCV specificity and immunochemical characteristics of the immunoglobulins involved in cryoprecipitation. METHODS Sera from 50 consecutive patients with chronic HCV infection (RNA positive) were screened for the presence of cryoglobulins. The two major components of cryoprecipitates, IgM rheumatoid factors and IgG, were separated by high performance liquid chromatography and analyzed for immunochemical composition by immunoblotting and antibody specificity by ELISA and immunoblotting using recombinant HCV proteins and synthetic peptides as antigens. RESULTS Cryoprecipitates were observed in 27 patients and characterized by immunofixation: 13 (48%) were classified as type II and 14 (52%) as type III. Monoclonal immunoglobulins were detected by immunoblotting in 20 cryoprecipitates: IgM in 14 samples and IgG in 14, with a clear preponderance of IgG3 (12/14). Specificity studies on sera and purified IgM and IgG fractions from cryoprecipitates revealed enrichment in cryoglobulins, predominantly polyclonal IgG1, reactive with the HCV structural proteins, whereas specificities for nonstructural viral proteins were relatively less represented compared to whole serum. No restricted pattern of fine specificity was observed. IgG3 subclass was apparently not involved in HCV nucleoprotein binding. CONCLUSIONS Our findings do not support a direct link between monoclonal cryoglobulins and immune response to HCV According to the proposed pathogenetic model, HCV infection can induce the formation of cryoprecipitable rheumatoid factors, sustain their production, and eventually lead to monoclonal B-cell expansion through several cooperative mechanisms.

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin THE CRUCIAL ROLE OF FIBRILLAR SEEDS

The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils.

Use of an Anti‐Idiotypic Monoclonal Antibody in Studying Amyloidogenic Light Chains in Cells, Urine and Fibrils: Pathophysiology and Clinical Implications

A monoclonal anti‐idiotype antibody (IgG1k MoAb 3B11D4) raised against the amyloidogenic DEPλ chain dimer binds a conformational idiotope also present on the monoclonal DEP IgA immunoglobulin. MoAb 3B11D4 does not recognize the reduced and alkylated λ chain monomers, nor the 15‐17‐kDa fibrillar light chain fragments which have the same N‐terminal sequence of the urinary light chains. The lack of about 70 ammo acid residues ofthe C terminal of the protein prevents the formation of the self‐limiting dimer and may facilitate the deposition of the fragments into amyloid fibrils. MoAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning experiments demonstrate that this antibody has the capability to selectively eliminate the idiotvpe positive cells from peripheral blood. Antibodies with these characteristics could become a useful tool for better understanding the pathogenesis of the disease and for new therapeutic options.

Identification of specific plasma proteins determining the agarose gel electrophoresis by the immunosubtraction technique.

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.

Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins.

Abstract 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.

Application of Monoclonal Anti-idiotypes in the Study of AL Amyloidosis: Therapeutic Implications

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 patients 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.