Synergy of combined Doxycycline/TUDCA treatment in lowering Transthyretin deposition and associated biomarkers: studies in FAP mouse models

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. We had previously shown that doxycycline administered for 3 months at 40 mg/Kg/ml in the drinking water, was capable of removing TTR amyloid deposits present in stomachs of old TTR-V30M transgenic mice; the removal was accompanied by a decrease in extracellular matrix remodeling proteins that accompany fibrillar deposition, but not of non-fibrillar TTR deposition and/or markers associated with pre-fibrillar deposits. On the other hand, Tauroursodeoxycholic acid (TUDCA), a biliary acid, administrated to the same mouse model was shown to be effective at lowering deposited non-fibrillar TTR, as well as the levels of markers associated with pre-fibrillar TTR, but only at young ages

The lung in amyloidosis

Amyloidosis is a disorder caused by misfolding of autologous protein and its extracellular deposition as fibrils, resulting in vital organ dysfunction and eventually death. Pulmonary amyloidosis may be localised or part of systemic amyloidosis.Pulmonary interstitial amyloidosis is symptomatic only if the amyloid deposits severely affect gas exchange alveolar structure, thus resulting in serious respiratory impairment. Localised parenchymal involvement may be present as nodular amyloidosis or as amyloid deposits associated with localised lymphomas. Finally, tracheobronchial amyloidosis, which is usually not associated with evident clonal proliferation, may result in airway stenosis.Because the treatment options for amyloidosis are dependent on the fibril protein type, the workup of all new cases should include accurate determination of the amyloid protein. Most cases are asymptomatic and need only a careful follow-up. Diffuse alveolar-septal amyloidosis is treated according to the underlying systemic amyloidosis. Nodular pulmonary amyloidosis is usually localised, conservative excision is usually curative and the long-term prognosis is excellent. Tracheobronchial amyloidosis is usually treated with bronchoscopic interventions or external beam radiation therapy

Certification of C-reactive Protein in Reference Material ERM-DA472/IFCC

The production and certification of ERM-DA472/IFCC, a new reference material certified for C-reactive protein (CRP), is described. ERM-DA472/IFCC was characterised using the reference material ERM-DA470 as calibrant. This achieved using a value transfer protocol that can be considered as a reference procedure. the principles used to measure the CRP concentration were immunonephelometry and immunoturbidimetry. The measurements were performed with different platform/reagent combinations (Abbott, Beckmann Immage, BN II, different Hitachi instruments, and Olympus AU640). In total 8 laboratories participated in the value assignment. The certified CRP mass concentration is 41.8 mg/L, the expanded uncertainty (k = 2) 2.5 mg/L.JRC.D.2-Reference material

The certification of mass concentration of Beta-2-microglobulin in human serum: ERM-DA470k/IFCC

This report describes the additional certification of the mass concentration of Beta-2-microglobulin (B2M) in ERM-DA470k/IFCC, a human serum material. The material was certified following ISO Guide 34:2009. The material was released in 2008 and was certified for the mass concentration of 12 proteins in human serum. A full description of the processing steps can be found in the original report. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-unit homogeneity was estimated to determine the minimum sample intake. The material was characterised by an intercomparison among laboratories of demonstrated competence and adhering to ISO/IEC 17025. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity, to instability and to characterisation. The material is intended for the calibration of immunoassay-based in-vitro diagnostic devices or control products for the proteins certified. As for any calibrator it should be verified that it is commutable. The material is produced in a similar manner as ERM-DA470, the use of which has led to a significant reduction in the between-method and between-laboratory variation for the proteins certified (B2M was not certified in this material) [ , ]. It was verified during the value assignment procedure that there were no significant matrix effects, and that different methods produced consistent results. However, when the material is used as a calibrator, the commutability should be verified for the particular assay concerned. The Certified Reference Material (CRM) is available in the lyophilised form of a 1.0 mL portion of serum with additives (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium azide, benzamidine hydrochloride, sodium chloride and aprotinin). The material is kept under nitrogen gas in threaded glass bottles with rubber stoppers and polypropylene screw caps. The water mass fraction of the sample is (4.3 ± 0.6) mg/g. The lyophilised powder has to be reconstituted with (1.00 ± 0.01) g of distilled water. The minimum amount of reconstituted sample to be used is 2 µL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Effects of tafamidis on transthyretin stabilization and clinical outcomes in patients with non-Val30Met transthyretin amyloidosis

This phase II, open-label, single-treatment arm study evaluated the pharmacodynamics, efficacy, and safety of tafamidis in patients with non-Val30Met transthyretin (TTR) amyloidosis. Twenty-one patients with eight different non-Val30Met mutations received 20 mg QD of tafamidis meglumine for 12 months. The primary outcome, TTR stabilization at Week 6, was achieved in 18 (94.7 %) of 19 patients with evaluable data. TTR was stabilized in 100 % of patients with non-missing data at Months 6 (n = 18) and 12 (n = 17). Exploratory efficacy measures demonstrated some worsening of neurological function. However, health-related quality of life, cardiac biomarker N-terminal pro-hormone brain natriuretic peptide, echocardiographic parameters, and modified body mass index did not demonstrate clinically relevant worsening during the 12 months of treatment. Tafamidis was well tolerated. In conclusion, our findings suggest that tafamidis 20 mg QD effectively stabilized TTR associated with several non-Val30Met variants

A novel approach for the purification and proteomic analysis of pathogenic immunglobulin 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

Proposed Cardiac End Points for Clinical Trials in Immunoglobulin Light Chain Amyloidosis: Report From the Amyloidosis Forum Cardiac Working Group

Immunoglobulin light chain amyloidosis is a rare, multisystemic, phenotypically heterogenous disease affecting cardiovascular, renal, neurological, and gastrointestinal systems to varying degrees. Its underlying cause is a plasma cell dyscrasia characterized by misfolding of monoclonal immunoglobulin light chains which leads to aggregation and deposition of insoluble amyloid fibrils in target organs. Prognosis is primarily dependent on extent of cardiac involvement and depth of hematologic response to treatment. To facilitate development of new therapies, a public-private partnership was formed between the nonprofit Amyloidosis Research Consortium and the US Food and Drug Administration Center for Drug Evaluation and Research. In 2020, the Amyloidosis Forum launched an initiative to identify novel/composite end points and analytic strategies to expedite clinical trials for development of new therapies for the primary hematologic disorder and organ system manifestations. Specialized working groups identified organ-specific end points; additional working groups reviewed health-related quality of life measures and statistical approaches to data analysis. Each working group comprised amyloidosis experts, patient representatives, statisticians, and representatives from the Food and Drug Administration, the UK Medicines and Healthcare Products Regulatory Agency, and pharmaceutical companies. This review summarizes the proceedings and recommendations of the Cardiac Working Group. Using a modified Delphi method, the group identified, reviewed, and prioritized cardiac end points relevant to immunoglobulin light chain amyloidosis in the context of an antiplasma cell therapy. Prioritized cardiovascular end points included overall survival, hospitalization, N-terminal pro-B-type natriuretic peptide level, 6-minute walk test, Kansas City Cardiac Questionnaire, and cardiac deterioration progression-free survival. These recommended components will be further explored through evaluation of clinical trial datasets and formal guidance from regulatory authorities

The red cell distribution width (RDW): value and role in preterm, IUGR (intrauterine growth restricted), full-term infants.

To measure the red cell distribution width (RDW) ranges at birth and to evaluate potential association with typical neonatal diseases: patent of the ductus arteriousus (PDA), bronchopulmonary dysplasia (BPD), and late-onset sepsis (LOS) mortality.Forty-six full-term, 41 preterm, and 35 intrauterine growth restricted (IUGR) infants participated in this retrospective, observational study. RDW was measured before 3 days of life (T0) in all infants, and at first month of life (T1) in preterm/IURG patients.RDW% mean (standard deviation) at T0 was: 15.65 (1.18) in full-term newborns; 17.7 (2.06) in preterm; 17.45 (1.81) in IUGR. A negative correlation (r = -0.51; P0.001) between RDW and gestational age was found. RDW at T1 was: 17.25 (2.19) in the preterm group; 17.37 (2.56) in IUGR group. Fourteen preterm infants reported: 12 PDA, 5 LOS, 4 BPD, and 3 died; 10 IUGR infants had: 4 PDA, 6 LOS, 3 BPD, and 1 died. RDW of IUGR infants suffering from those pathologies was not statistically different compared with unaffected infants, while preterm newborns with pathologies reported higher RDW: PDA vs. PDA absent: P = 0.008 at T0; P0.002 at T1. BPD vs. BPD absent: P0.005 at T1. LOS vs. LOS absent: P0.005 at T0. RDW in preterm/IUGR population was associated with early mortality, T0: dead 21.2 (2.7) vs. alive 16.7 (1.7), P0.0001.RDW and gestational age at birth were negatively correlated. High RDW resulted to be an indication of risk for critical newborns. This parameter can be inexpensively and routinely verified and further studies are required to confirm its prognostic role in neonatal pathologies

Diagnosis, Prognosis, and Therapy of Transthyretin Amyloidosis

AbstractTransthyretin amyloidosis is a fatal disorder that is characterized primarily by progressive neuropathy and cardiomyopathy. It occurs in both a mutant form (with autosomal dominant inheritance) and a wild-type form (with predominant cardiac involvement). This article guides clinicians as to when the disease should be suspected, describes the appropriate diagnostic evaluation for those with known or suspected amyloidosis, and reviews the interventions currently available for affected patients