Circulating free light chain measurement in the diagnosis, prognostic assessment and evaluation of response of AL amyloidosis: comparison of Freelite and N latex FLC assays.

BACKGROUND: The measurement of circulating free light chain (FLC) is essential in the diagnosis, prognostic stratification and evaluation of response to therapy in light chain (AL) amyloidosis. For more than 10 years, this has been done with an immunonephelometric assay based on polyclonal antibodies (Freelite), and cutoffs for staging and response assessment have been validated with this method. Recently, a new assay based on monoclonal antibodies (N latex FLC) has been marketed in Europe. METHODS: We evaluated and compared the clinical performance of the two assays in 426 patients with newly diagnosed AL amyloidosis. RESULTS: We found suboptimal agreement between the two methods, with differences between values obtained with the Freelite and N latex FLC assays increasing with the concentration of clonal FLC. The diagnostic sensitivity of the Freelite (82%) and N latex FLC (84%) assays was similar, and both improved to 98% in combination with serum and urine immunofixation. The concentration of FLC measured with both methods had prognostic significance. Less pronounced decreases in FLC best predicted improved survival with the N latex FLC assay (33% vs. 50%), and there was poor concordance (84%) in discrimination of responders. CONCLUSIONS: The two assays have similar diagnostic and prognostic performance. However, they are not interchangeable, and follow-up should be done with either one. New response criteria are needed for the N latex FLC assay.

A Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart amyloidosis.

Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies.

Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis.

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.

A patient with AL amyloidosis with negative free light chain results.

The detection and quantification of amyloidogenic monoclonal light chains are necessary for the diagnosis and evaluation of response to treatment in AL amyloidosis. However, the amyloid clone is often small and difficult to detect. We report the case of a 68-year-old man who was referred to our Center in April 2013 after syncope and the identification of left ventricular hypertrophy at echocardiography, suspected for amyloidosis. A commercial agarose gel electrophoresis immunofixation (IFE) did not reveal monoclonal components in serum and urine. The κ serum free light chain (FLC) concentration was 21.5 mg/L, λ 33 mg/L (κ/λ ratio 0.65), NT-proBNP 9074 ng/L (u.r.l. <332 ng/L) and an echocardiogram confirmed characteristic features of amyloidosis. The abdominal fat aspiration was positive and the amyloid typing by immune-electron microscopy revealed λ light chains deposits. A high-resolution (hr) IFE of serum and urine showed a faint monoclonal λ component in the urine. A bone marrow biopsy showed 8% plasma cells (BMPC) and a kappa/lambda light-chain restriction with λ light chain on immunofluorescence. The diagnosis of AL (λ) amyloidosis with cardiac involvement was made. In May 2013, patient was started on cyclophosphamide, bortezomib and dexamethasone. After six cycles, serum and urine hr-IFE were negative, the bone marrow biopsy showed 3% BMPC without light chain restriction by immunofluorescence, and a decrease of NT-proBNP was observed (5802 ng/L).Thus, treatment was discontinued. In this patient the amyloid clone could be detected only by in house hr-IFE of urine and bone marrow examination. The detection of the small dangerous amyloidogenic clone should be pursued with a combination of high-sensitivity techniques, including assessment of BMPC clonality. Studies of novel tools, such as mass spectrometry on serum and next-generation flow cytometry analysis of the bone marrow, for detecting plasma cell clones in AL amyloidosis and other monoclonal light chain-related disorders are warranted.

The impact of renal function on the clinical performance of FLC measurement in AL amyloidosis.

BACKGROUND: The measurement of circulating free light chains (FLC) is of utmost importance in immunoglobulin light chain (AL) amyloidosis, being a fundamental part of the diagnostic workup, prognostic stratification and assessment of response to therapy. Renal failure is a common feature of AL amyloidosis and can considerably affect the concentration of FLC. METHODS: We assessed the impact of renal failure on the clinical performance of the Freelite assay in 982 consecutive, newly diagnosed patients with AL amyloidosis, 822 with estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2, and 160 with eGFR <30 mL/min/1.73 m2. RESULTS: The diagnostic sensitivity of the κ/λ FLC ratio was lower for λ amyloidogenic FLC in patients with renal failure (81% vs. 60%, p<0.001) and the FLC concentration had no independent prognostic significance in patients with severe renal dysfunction. However, FLC response to chemotherapy could still discriminate patients with better outcome. CONCLUSIONS: Renal failure is a relevant interference factor when using the Freelite assay for the identification of the amyloidogenic light chain and for prognostic assessment in patients with AL amyloidosis and renal failure.

Reliable typing of systemic amyloidoses through proteomic analysis of subcutaneous adipose tissue.

Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.

Shotgun protein profile of human adipose tissue and its changes in relation to systemic amyloidoses.

In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases.

Surface Nanotexturing of Boron-Doped Diamond Films by Ultrashort Laser Pulses.

Polycrystalline boron-doped diamond (BDD) films were surface nanotextured by femtosecond pulsed laser irradiation (100 fs duration, 800 nm wavelength, 1.44 J cm-2 single pulse fluence) to analyse the evolution of induced alterations on the surface morphology and structural properties. The aim was to identify the occurrence of laser-induced periodic surface structures (LIPSS) as a function of the number of pulses released on the unit area. Micro-Raman spectroscopy pointed out an increase in the graphite surface content of the films following the laser irradiation due to the formation of ordered carbon sites with respect to the pristine sample. SEM and AFM surface morphology studies allowed the determination of two different types of surface patterning: narrow but highly irregular ripples without a definite spatial periodicity or long-range order for irradiations with relatively low accumulated fluences (<14.4 J cm-2) and coarse but highly regular LIPSS with a spatial periodicity of approximately 630 nm ± 30 nm for higher fluences up to 230.4 J cm-2.

Etching Kinetics of Nanodiamond Seeds in the Early Stages of CVD Diamond Growth.

We present an experimental study on the etching of detonation nanodiamond (DND) seeds during typical microwave chemical vapor deposition (MWCVD) conditions leading to ultra-thin diamond film formation, which is fundamental for many technological applications. The temporal evolution of the surface density of seeds on the Si(100) substrate has been assessed by scanning electron microscopy (SEM). The resulting kinetics have been explained in the framework of a model based on the effect of the particle size, according to the Young-Laplace equation, on both chemical potential of carbon atoms in DND and activation energy of the reaction with atomic hydrogen. The model describes the experimental kinetics of seeds' disappearance by assuming that nanodiamond particles with a size smaller than a "critical radius," r*, are etched away while those greater than r* can grow. Finally, the model allows to estimate the rate coefficients for growth and etching from the experimental kinetics.

Frenkel-Poole Mechanism Unveils Black Diamond as Quasi-Epsilon-Near-Zero Surface.

A recent innovation in diamond technology has been the development of the "black diamond" (BD), a material with very high optical absorption generated by processing the diamond surface with a femtosecond laser. In this work, we investigate the optical behavior of the BD samples to prove a near to zero dielectric permittivity in the high electric field condition, where the Frenkel-Poole (FP) effect takes place. Zero-epsilon materials (ENZ), which represent a singularity in optical materials, are expected to lead to remarkable developments in the fields of integrated photonic devices and optical interconnections. Such a result opens the route to the development of BD-based, novel, functional photonic devices.

A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.

An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms.

Insights into the Thermally Activated Cyclization Mechanism in a Linear Phenylalanine-Alanine Dipeptide.

Dipeptides, the prototype peptides, exist in both linear (l-) and cyclo (c-) structures. Since the first mass spectrometry experiments, it has been observed that some l-structures may turn into the cyclo ones, likely via a temperature-induced process. In this work, combining several different experimental techniques (mass spectrometry, infrared and Raman spectroscopy, and thermogravimetric analysis) with tight-binding and ab initio simulations, we provide evidence that, in the case of l-phenylalanyl-l-alanine, an irreversible cyclization mechanism, catalyzed by water and driven by temperature, occurs in the condensed phase. This process can be considered as a very efficient strategy to improve dipeptide stability by turning the comparatively fragile linear structure into the robust and more stable cyclic one. This mechanism may have played a role in prebiotic chemistry and can be further exploited in the preparation of nanomaterials and drugs.

Mn-Containing Bioactive Glass-Ceramics: BMP-2-Mimetic Peptide Covalent Grafting Boosts Human-Osteoblast Proliferation and Mineral Deposition.

The addition of Mn in bioceramic formulation is gaining interest in the field of bone implants. Mn activates human osteoblast (h-osteoblast) integrins, enhancing cell proliferation with a dose-dependent effect, whereas Mn-enriched glasses induce inhibition of Gram-negative or Gram-positive bacteria and fungi. In an effort to further optimize Mn-containing scaffolds' beneficial interaction with h-osteoblasts, a selective and specific covalent functionalization with a bioactive peptide was carried out. The anchoring of a peptide, mapped on the BMP-2 wrist epitope, to the scaffold was performed by a reaction between an aldehyde group of the peptide and the aminic groups of silanized Mn-containing bioceramic. SEM-EDX, FT-IR, and Raman studies confirmed the presence of the peptide grafted onto the scaffold. In in vitro assays, a significant improvement in h-osteoblast proliferation, gene expression, and calcium salt deposition after 7 days was detected in the functionalized Mn-containing bioceramic compared to the controls.

Charge Transport Mechanisms of Black Diamond at Cryogenic Temperatures.

Black diamond is an emerging material for solar applications. The femtosecond laser surface treatment of pristine transparent diamond allows the solar absorptance to be increased to values greater than 90% from semi-transparency conditions. In addition, the defects introduced by fs-laser treatment strongly increase the diamond surface electrical conductivity and a very-low activation energy is observed at room temperature. In this work, the investigation of electronic transport mechanisms of a fs-laser nanotextured diamond surface is reported. The charge transport was studied down to cryogenic temperatures, in the 30−300 K range. The samples show an activation energy of a few tens of meV in the highest temperature interval and for T < 50 K, the activation energy diminishes to a few meV. Moreover, thanks to fast cycles of measurement, we noticed that the black-diamond samples also seem to show a behavior close to ferromagnetic materials, suggesting electron spin influence over the transport properties. The mentioned properties open a new perspective in designing novel diamond-based biosensors and a deep knowledge of the charge-carrier transport in black diamond becomes fundamental.

Enhanced and Selective Absorption of Molybdenum Nanostructured Surfaces for Concentrated Solar Energy Applications.

Surfaces of commercial molybdenum (Mo) plates have been textured by fs-laser treatments with the aim to form low-cost and efficient solar absorbers and substrates for thermionic cathodes in Concentrated Solar Power conversion devices. Morphological (SEM and AFM), optical (spectrophotometry), and structural (Raman spectroscopy) properties of the samples treated at different laser fluences (from 1.8 to 14 J/cm2) have been characterized after the laser treatments and also following long thermal annealing for simulating the operating conditions of thermionic converters. A significant improvement of the solar absorptance and selectivity, with a maximum value of about four times higher than the pristine sample at a temperature of 800 K, has been detected for sample surfaces treated at intermediate fluences. The effects observed have been related to the light trapping capability of the laser-induced nanotexturing, whereas a low selectivity, together with a high absorptance, could be revealed when the highest laser fluence was employed due to a significant presence of oxide species. The ageing process confirms the performance improvement shown when treated samples are used as solar absorbers, even though, due to chemical modification occurring at the surface, a decrease of the solar absorptance takes place. Interestingly, the sample showing the highest quantity of oxides preserves more efficiently the laser texturing. The observation of this behaviour allows to extend the applicability of the laser treatments since, by further nanostructuring of the Mo oxides, it could be beneficial also for sensing applications.

Femtosecond-Laser Nanostructuring of Black Diamond Films under Different Gas Environments.

Irradiation of diamond with femtosecond (fs) laser pulses in ultra-high vacuum (UHV) conditions results in the formation of surface periodic nanostructures able to strongly interact with visible and infrared light. As a result, native transparent diamond turns into a completely different material, namely "black" diamond, with outstanding absorptance properties in the solar radiation wavelength range, which can be efficiently exploited in innovative solar energy converters. Of course, even if extremely effective, the use of UHV strongly complicates the fabrication process. In this work, in order to pave the way to an easier and more cost-effective manufacturing workflow of black diamond, we demonstrate that it is possible to ensure the same optical properties as those of UHV-fabricated films by performing an fs-laser nanostructuring at ambient conditions (i.e., room temperature and atmospheric pressure) under a constant He flow, as inferred from the combined use of scanning electron microscopy, Raman spectroscopy, and spectrophotometry analysis. Conversely, if the laser treatment is performed under a compressed air flow, or a N2 flow, the optical properties of black diamond films are not comparable to those of their UHV-fabricated counterparts.

Nanostructured sp2-carbon infiltration of mesoporous silicon layers.

The preparation of composite layers made of porous silicon (PS) infiltrated with nanostructured carbon is reported. These composite layers were obtained by chemical vapor infiltration (CVI) of mesoporous silicon under process conditions normally employed to grow diamond films by Hot Filament Chemical Vapour Deposition (HFCVD). Micro-Raman spectroscopy and Field Emission Gun Scanning Electron Microscopy (FEG-SEM) techniques showed that diamond nucleation density was very low whilst sp2 carbon permeated completely, even after 1 h deposition, the thickness of the PS layers that preserved their mesoporous columnar structure.

Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the levels of intracellular proteins in human heart cells.

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells.

Investigating heart-specific toxicity of amyloidogenic immunoglobulin light chains: A lesson from C. elegans.

Abnormalities in protein folding are involved in many localized and systemic diseases, all of which are characterized by insoluble amyloid formation and deposition. In immunoglobulin light chain (LC) amyloidosis, the most frequent systemic form of amyloidosis, the amyloid involvement of the heart dictates the prognosis and the elucidation of the mechanism of heart targeting and toxicity is essential for designing and testing new effective treatments. To this end, the availability of an appropriate animal model is crucial. We recently described the use of C. elegans as an innovative experimental system to investigate in vivo the pathogenic effects of monoclonal LC. This idea stems from the knowledge that the worm's pharynx is an "ancestral heart" with the additional ability to recognize stressor compounds. The feeding of worms with LC purified from patients suffering from cardiomyopathy, selectively and permanently impaired the pharyngeal function. This irreversible damage resulted in time, in a significant reduction in the lifespan of worms. We also reported that the ability of LC to generate reactive oxygen species was associated with their toxic effects and was counteracted by anti-oxidant compounds. This new nematode-based assay represents a promising model for elucidating the heart-specific toxicity of LC and for a rapid screening of new therapeutic strategies.