Periacetabular osteotomy for hip dysplasia treatment through a mini-invasive technique. Our results at mid-term in 131 cases. / Osteotomía periacetabular en el tratamiento de displasia de cadera mediante técnica mini-invasiva. Nuestros resultados a medio plazo en 131 casos.

BACKGROUND AND OBJECTIVE: Periacetabular osteotomy (PAO) is an accepted and worldwide technique recognized for residual dysplasia treatment and even in unstable hips with limited acetabular coverage. The aim of this study is to analyse the functional, radiological and complication results in patients treated with mini-invasive PAO. MATERIAL AND METHODS: We performed a retrospective study in which we analysed 131 cases undergoing mini-invasive PAO at our centre. The degree of joint degeneration was evaluated with Tönnis scale, Wiberg angle, acetabular index (AI), anterior coverage angle (AC), joint space, complications and functional outcome with the Non-Arthritic Hip Score (NAHS) were analysed preoperatively and at the end of follow-up. RESULTS: The average age was 32.3±9.5 (SD) years, 102 (77.9%) were female and 29 (22.1%) were male. 7.7±2.8 (SD) years follow up. The radiological parameters improved between the pre-surgical phase and the end of follow-up, Wiberg angle+18.5° (18.3° versus 36.8°, 95% CI 17.3 to 19.7), AC angle+13.5° (26.2° versus 39.7°, 95%CI 11.6 to 15.4) and the AI -11.1° (19.5° versus 8.4°; 95%CI -12.1 to -10,1). In addition, the functional results, with the NAHS scale, improved+31.3 points (60.7 pre-surgical versus 92 at the end of follow-up, 95% CI 28.7 to 33.8). The most common complication was transient lateral femoral cutaneous nerve hypoaesthesia in 10 cases (7%). CONCLUSION: The mini-invasive PAO approach is a reproducible technique, it allows restoration of acetabular coverage and provides an improvement in functional scales as confirmed by our series.

Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.

Structural characterization of kappa II Inc, a new amyloid immunoglobulin.

Light chain Inc, obtained from a patient with amyloid arthropathy, has an Mr of 23,550 and consists of 219 amino acid residues. The complete primary structure of its variable domain has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide digestion, and by partially sequencing the intact protein. Although closely related to protein of the V kappa II subgroup, light chain Inc differs from its counterpart by the replacement of some invariant residues in its variable domain. By comparing its sequence with that of the nonamyloid kappa II Nim, a different distribution of some polar and apolar amino acid residues through the molecule is evidenced. A computer graphic analysis shows that some of the replaced amino acid residues cannot be readily accommodated in the known three-dimensional structure of the immunoglobulin light chains.

Amino acid sequence of k Sci, the Bence Jones protein isolated from a patient with light chain deposition disease.

Light chain Sci was isolated from the urine of a patient affected by light chain deposition disease with an apparent exclusive localization to the kidney. Sci protein is an intact light chain: it consists of 214 amino acid residues and has an Mr of 23.65. Its complete primary structure has been determined by sequence analysis of the corresponding tryptic peptides and by partially sequencing the intact protein. Sequence comparison shows that Sci protein is strictly related to the light chains of kIIIa family (88% structural identity) which are usually expressed in autoimmune rheumatoid syndromes. Computer graphics model suggests a perturbation in k Sci three-dimensional structure due to the unusual replacement of residues 53 and 77.

Structural and functional characterization of three human immunoglobulin kappa light chains with different pathological implications.

The structural properties of three immunoglobulins light chains: kappa SCI, responsible for light chain deposition disease (Bellotti, V., Stoppini, M., Merlini, G., Zapponi, M.C., Meloni, M.L., Banfi, G. and Ferri, G. (1991) Biochim. Biophys. Acta 1097, 177-182), k INC responsible for light chain amyloidosis (Ferri, G., Stoppini, M., Iadarola, P., Bellotti, V. and Merlini, G. (1989) Biochim. Biophys. Acta 995, 103-108) and the non-pathogenic kappa MOS were analyzed by fluorescence spectroscopy and circular dichroism. Comparative evaluation of the data shows that SCI and MOS have similar stability under different conditions, while the amyloid k INC behaves as a very unstable protein. As calculated from the GdnHCl curves, the midpoint of unfolding transition was 1.35 M for SCI, 1.20 M for MOS and 0.1 M for INC. Analysis of CD spectra evidences that the three proteins conserve their conformation in the range of pH 4-8. Change in temperature at pH 4.0 produces the premature transition of INC (Tm 40 degrees C) with respect to SCI and MOS (Tm 50 degrees C). At this pH both the pathological SCI and INC light chains aggregate at a temperature of 20 degrees C lower than the normal counterpart. The specific kidney deposition of kappa SCI has been evidenced after injection of the 125I labelled light chain into mice. No deposition was detectable in the case of INC and MOS.

Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin.

beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation.

beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation.

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure.

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.

Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

Characterization of apo(a) polymorphism by a modified immunoblotting technique in an Italian population sample.

Apo(a), the specific lipoprotein(a) (Lp(a)) apolipoprotein, is characterized by different isoforms (from 6 to 11 on SDS-PAGE) encoded by a system of autosomal codominant alleles. Electrophoresis on agarose gel displays a better resolving power than SDS-PAGE (a larger number of apo(a) isoforms is detected). The aim of this work was to set up a simple technique that uses a capillary blotting apparatus and a polyvinylidene difluoride membrane for protein transfer. We tested an Italian population sample of 202 healthy subjects (123 men and 79 women) and we detected 22 apo(a) isoforms varying from 280 to 775 kDa. In our sample, 135 subjects (66.5%) had a single-band phenotype, 64 (31.7%) had a double-band phenotype and 3 subjects (1.5%) had no detectable bands ('null' phenotype). This simple and reproducible technique could be applied in the genetic screening of apo(a) polymorphisms and for clinical investigations of the risk of developing cardiovascular diseases.

Immunochemical characteristics of a particular cryoglobulin. A new cryoglobulin subgroup?

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.

Immunological reactivity of serum ferritin in patients with malignancy.

Serum ferritin has been suggested as a tumor marker in the diagnosis of certain malignancies and for following the activity or dissemination of the malignant process. Since neoplastic tissues generally contain more acidic isoferritins than their normal tissue counterparts, it has also been suggested that the specific assay of such isoferritins in serum may be of particular value in the diagnosis of malignancy. In this work, we have evaluated ferritin concentration in the serum of normal subjects and patients with acute nonlymphocytic leukemia, Hodgkin's disease, breast cancer and lung cancer by simultaneously using three different immunoassays: an immunoradiometric assay based on polyclonal antibodies against human liver (basic, L-subunit rich) ferritin, a radioimmunoassay based on polyclonad antibodies against HeLa cell (acidic, H-subunit rich) ferritin, and an immunoradiometric assay based on the monoclonal antibody 2A4 raised against human heart (acidic, H-subunit rich) ferritin. Most of the patients studied had increased values for liver-type ferritin in the absence of increased iron stores. Binding of serum ferritin to concanavalin A did not prove to be useful in distinguishing a tumor-specific basic isoferritin. The HeLa ferritin assay was found to be less specific than the heart ferritin assay in the detection of acidic isoferritins, and did not provide any advantage over the liver assay in detecting the increased levels of serum ferritin associated with malignant disease. Heart-type ferritin was found in one-fifth of normal sera and 64% of sera from patients with malignancy. Values were very low compared with those for basic ferritin, ranging from less than 0.1 to 17% of total serum ferritin (geometric mean value 1.3%) in patients with malignancy. These findings indicate that at present there is little application for serum ferritin immunoassays based on antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of malignant disease. This seems to be due to the presence in human serum of biding factors which are responsible for the rapid clearance of acidic isoferritins from the circulation. The serum concentration of basic ferritin, however, can be useful in the diagnosis and management of some malignancies, and it is possible that studies on cell isoferritins can be important in biologic monitoring of neoplastic disorders. It should also be noted that the increased levels of serum ferritin found in patients with malignancy can exert adverse effects on the host immune response and perhaps an inhibitory effect on hematopoiesis.

Osteotomía periacetabular en el tratamiento de displasia de cadera mediante técnica mini-invasiva. Nuestros resultados a medio plazo en 131 casos / Periacetabular osteotomy for hip dysplasia treatment through a mini-invasive technique. Our results at mid-term in 131 cases

ANTECEDENTES Y OBJETIVO: La osteotomía periacetabular (OPA) es una técnica utilizada para el tratamiento de la displasia residual, incluso en caderas inestables con cobertura acetabular limitada. El objetivo de este estudio es analizar los resultados funcionales, radiológicos y las complicaciones en pacientes tratados mediante OPA mini-invasiva. MATERIALES Y MÉTODOS: Estudio retrospectivo que analiza 131 casos intervenidos con OPA en nuestro centro. Se determinó de forma prequirúrgica y al final del seguimiento el grado de degeneración articular con la escala de Tönnis, el ángulo de Wiberg, el índice acetabular, el ángulo de cobertura anterior, el espacio articular, las posibles complicaciones y el resultado funcional mediante la escala Non-Arthritic Hip Score. RESULTADOS: La edad media de 32,3±9,5 (DE) años, 102 (77,9%) fueron mujeres y 29 (22,1%) fueron hombres. El seguimiento fue de 7,7±2,8 (DE) años. Se obtuvo una mejora en los parámetros radiológicos entre el momento prequirúrgico y al final del seguimiento, ángulo de Wiberg de+18,5° (18,3° versus 36,8°, IC 95%: 17,3 a 19,7), ángulo de cobertura anterior de+13,5° (26,2° versus 39,7°, IC 95%: 11,6 a 15,4) y el índice acetabular de -11,1° (19,5° versus 8,4°; IC 95%: -12,1 a -10,1). Además, los resultados funcionales con la escala Non-Arthritic Hip Score mejoraron en+31,3 puntos (60,7 prequirúrgico versus 92 último seguimiento posquirúrgico; IC 95%: 28,7 a 33,8). La complicación más frecuente fue la disestesia transitoria del nervio fémoro-cutáneo lateral en 10 casos (7%). CONCLUSIÓN: La osteotomía periacetabular mediante el abordaje mini-invasivo es una técnica reproducible, permite restaurar la cobertura acetabular y proporciona una mejora en las escalas funcionales según confirma nuestra serie

BACKGROUND AND OBJECTIVE: Periacetabular osteotomy (PAO) is an accepted and worldwide technique recognized for residual dysplasia treatment and even in unstable hips with limited acetabular coverage. The aim of this study is to analyse the functional, radiological and complication results in patients treated with mini-invasive PAO. MATERIAL AND METHODS: We performed a retrospective study in which we analysed 131 cases undergoing mini-invasive PAO at our centre. The degree of joint degeneration was evaluated with Tönnis scale, Wiberg angle, acetabular index (AI), anterior coverage angle (AC), joint space, complications and functional outcome with the Non-Arthritic Hip Score (NAHS) were analysed preoperatively and at the end of follow-up. RESULTS: The average age was 32.3±9.5 (SD) years, 102 (77.9%) were female and 29 (22.1%) were male. 7.7±2.8 (SD) years follow up. The radiological parameters improved between the pre-surgical phase and the end of follow-up, Wiberg angle+18.5° (18.3° versus 36.8°, 95% CI 17.3 to 19.7), AC angle+13.5° (26.2° versus 39.7°, 95%CI 11.6 to 15.4) and the AI -11.1° (19.5° versus 8.4°; 95%CI -12.1 to -10,1). In addition, the functional results, with the NAHS scale, improved+31.3 points (60.7 pre-surgical versus 92 at the end of follow-up, 95% CI 28.7 to 33.8). The most common complication was transient lateral femoral cutaneous nerve hypoaesthesia in 10 cases (7%). CONCLUSION: The mini-invasive PAO approach is a reproducible technique, it allows restoration of acetabular coverage and provides an improvement in functional scales as confirmed by our series

Current concepts on the pathogenesis of systemic amyloidosis.

Amyloidosis is a pathological condition in which protein is deposited extracellularly in the form of insoluble fibrils that lead to organ dysfunction and death. Many different types of proteins are known to form amyloid and cause a heterogeneous array of clinical conditions. The unifying aspect of these conditions is the common structural entity resulting from the assembly of a primarily beta-structure protein into 5-10 nm wide non-branching insoluble fibrils displaying the characteristic green birefringence of bound Congo red dye when viewed under polarized light. Several factors contribute to amyloid assembly. Certain biophysical characteristics of the amyloidogenic precursor influence amyloidogenicity. Any mutation that sufficiently decreases protein stability favours the formation of a partially folded state under physiological conditions. This intermediate exposes other key sequence elements to the solvent, i.e. hydrophobic or charged residues that decrease solubility and promote aggregation and ultimately amyloid formation. In addition to primary protein structure, which confers a susceptibility to amyloid formation, other elements are probably important for the initiation, development and persistence of amyloid deposits: proteoglycans, amyloid P component, apolipoprotein E and others, most of which are normal constituents of basement membranes. The role of these factors in amyloidogenesis has been studied in two major systemic amyloidoses with prominent renal involvement: light-chain and beta-2-microglobulin amyloidosis. A detailed understanding of the molecular processes leading to amyloid deposition is required for the development of effective therapies.

Review: immunoglobulin light chain amyloidosis--the archetype of structural and pathogenic variability.

AL amyloidosis is caused by deposition in target tissue of amyloid fibrils constituted by monoclonal immunoglobulin light chains. The amyloidogenic plasma cells derive from a transformed memory B cell that can be identified by anti-idiotype monoclonal antibodies. Comparison of the primary structures of amyloidogenic and nonamyloidogenic light chains does not show any common structural motif in the amyloidogenic variants but reveals peculiar replacements which can destabilize the folding state. Reduced folding stability now appears to be a unifying property of amyloidogenic light chains. The tendency of these proteins to populate a partially unfolded intermediate state is a key event in the self-association that progresses to the formation of oligomers and fibrils. The mechanism of organ damage caused by AL amyloid deposition is not known, but clinical findings suggest that the process of amyloid fibril formation itself exerts tissue toxic effects independently of the amount of amyloid deposited. Since the disease is caused by the neoplastic expansion of the plasma cell population synthesizing the amyloidogenic light chains, the clone represents the prime therapeutic target of conventional chemotherapy and experimental immunotherapy. In common with other types of amyloidosis the therapeutic strategy can take advantage of drugs able to improve the reabsorption of the amyloid deposits or able to bind and stabilize the light chain in the native-like folded state.

Biological activity and pathological implications of misfolded proteins.

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, beta 2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies.

Dynamic of beta(2)-microglobulin fibril formation and reabsorption: the role of proteolysis.

Dialysis-related amyloidosis (DRA) is caused by the deposition, in target tissues, of beta(2)-microglobulin (beta(2)M) in fibrillar conformation. Several reports indicate that fibrillar beta(2)M is chemically heterogeneous and such heterogeneity is partially related to the presence of truncated species of the protein. In association with the full-length species, a beta(2)M isoform lacking six N-terminal residues is present in all the samples of our collection of ex vivo fibrils. The pattern of proteolytic cleavage in amyloidosis and in other diseases is completely different, as demonstrated by the absence in fibrillar beta(2)M of the cleavage at lysine 58, which is contrary to that described in rheumatoid arthritis and other diseases. The role of limited proteolysis of beta(2)M in the pathogenesis of the disease is uncertain. However, we have shown that the apparently minor modification of the intact protein, such as the removal of N-terminal hexapeptide, is capable of dramatically affecting its stability, protection from proteolytic digestion, and enhance its capacity to make in vitro amyloid fibrils. The structure, folding dynamic, and function of the truncated species of beta(2)M, peculiar of DRA, could shed new light on the mechanism of beta(2)M fibril formation and reabsorption.

New drug therapy of amyloidoses: resorption of AL-type deposits with 4'-iodo-4'-deoxydoxorubicin.

Amyloidosis caused by monoclonal Ig light chains (AL) is characterized by the tissue deposition of paraproteins as insoluble fibrils that leads to organ dysfunction and death. After serendipitous observation of its efficacy, the new anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX) was evaluated in eight patients with biopsy-proven AL and symptomatic organ involvement who received 1 to 6 administrations of I-DOX at dosages of 15 to 100 mg/m2. Five patients showed substantial clinical improvement concomitant with instrumental and physical evidence of response, and three patients presented objective evidence of amyloid resorption. The effects of I-DOX on amyloid deposits were not associated with cytotoxicity to the amyloidogenic clone. Five patients died of disease-related complications at 4 to 36 months; the remaining three are alive 29, 35, and 44 months after starting treatment. I-DOX caused short-lived granulocytopenia and minimal extra-hematologic side effects. The pharmacokinetics of I-DOX presented features exploitable for diagnosis in amyloidotic patients and documented the active metabolite in the cerebrospinal fluid. We conclude that I-DOX represents an important treatment option for subjects with AL amyloidosis and could be the prototype of a new class of drugs that interfere with and reverse the process of all types of amyloid deposition.