Early data on long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloidosis: a 2-year update from the open-label extension of the NEURO-TTR trial.

BACKGROUND AND PURPOSE: Hereditary transthyretin (hATTR) amyloidosis causes progressive polyneuropathy resulting from transthyretin (TTR) amyloid deposition throughout the body, including the peripheral nerves. The efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of TTR protein production, were demonstrated in the pivotal NEURO-TTR study in patients with hATTR polyneuropathy. Here, the long-term efficacy and safety of inotersen are assessed in an ongoing open-label extension (OLE) study. METHODS: Patients who completed NEURO-TTR were eligible to enroll in the OLE (NCT02175004). Efficacy assessments included the modified Neuropathy Impairment Score plus seven neurophysiological tests composite score (mNIS + 7), the Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) questionnaire total score and the Short-Form 36 Health Survey (SF-36) Physical Component Summary (PCS) score. Safety and tolerability were also assessed. RESULTS: Overall, 97% (135/139) of patients who completed NEURO-TTR enrolled in the OLE. Patients who received inotersen for 39 cumulative months in NEURO-TTR and the OLE continued to show benefit; patients who switched from placebo to inotersen in the OLE demonstrated improvement or stabilization of neurological disease progression by mNIS + 7, Norfolk QOL-DN and SF-36 PCS. No new safety concerns were identified. There was no evidence of increased risk for grade 4 thrombocytopenia or severe renal events with increased duration of inotersen exposure. CONCLUSION: Inotersen slowed disease progression and reduced deterioration of quality of life in patients with hATTR polyneuropathy. Early treatment with inotersen resulted in greater long-term disease stabilization than delayed initiation. Routine platelet and renal safety monitoring were effective; no new safety signals were observed.

Serum leptin, resistin, visfatin and adiponectin levels in tumor necrosis factor receptor-associated periodic syndrome (TRAPS).

OBJECTIVES: The aims of our study were to evaluate serum leptin, resistin, visfatin and adiponectin levels in patients with tumour necrosis factor receptor-associated periodic syndrome (TRAPS), in comparison to healthy controls, and to correlate their levels to parameters of disease activity and/or severity. METHODS: Serum leptin, resistin, visfatin and adiponectin levels were obtained from 14 TRAPS patients carrying mutations involving cysteine residues, from 16 TRAPS patients carrying other mutations, and from 16 healthy controls. Demographic, clinical and laboratory parameters, including amyloidosis were entered for each patient. Comparisons between groups as well as reciprocal comparisons have been evaluated. RESULTS: Serum leptin, resistin, visfatin and adiponectin did not significantly differ among the 3 groups. Patients carrying cysteine residues mutations showed lower visfatin serum levels than patients carrying other mutations (p<0.02). Serum leptin significantly correlated with the number of attacks/year (multiple R=0.32, multiple adjusted R2= 0.19, p <0.03). Serum adiponectin levels significantly correlated with the presence of amyloidosis (multiple R=0.79, multiple adjusted R2=0.57, p<0.03). Adiponectin values were a significant predictor for amyloidosis (AUC 0.75, 95 CI: 0.56-0.94, p<0.03), with a predicting cut-off value set at 23.16 pg/ml, the predictive positive value was 53.8%. Visfatin serum levels resulted respectively related to leptin (rs=0.42, r2=0.18, p<0.02) and to resistin (rs=0.57, r2=0.32, p<0.01) serum levels; whilst leptin and resistin serum levels did not reciprocally correlate. CONCLUSIONS: Although a prospective design study and larger cohort are mandatory, adipokines serum levels and their correlations with parameters of disease activity and/or severity seem to show a baseline pattern in TRAPS patients.

Severity in biliary peritonitis.

OBJECTIVE: To analyze clinical and laboratory findings in order to find variables predictive of severity of Biliary Peritonitis (BP). Patients and methods. Physical findings, course of illness, imaging and laboratory data were evaluated in 42 patients with BP, and statistically analysed to assess their prognostic significance. RESULTS: Serious illness and worse outcome were associated with: age ≥ 60 years (P=0.034), long time between onset of symptoms and treatment (P=0.025), fever > 38°C (P=0.009), WBC count > 17,000 cell/mm³ (P=0.043), diffuse abdominal pain (P=0.034), and infected bile (P=0.048). CONCLUSIONS: Most patients become severely ill due to supervening infection, while early bile drainage avoids serious complications. In addition, abdominal pain, fever and WBC count are also predictive of severity of BP.

Insulin-like growth factor-I (IGF-1), IGF-binding protein-3 (IGFBP-3) and mammographic features.

INTRODUCTION: The IGF system has recently been shown to play an important role in the regulation of breast tumor cell proliferation. However, also breast density is currently considered as the strongest breast cancer risk factor. It is not yet clear whether these factors are interrelated and if and how they are influenced by menopausal status. The purpose of this study was to examine the possible effects of IGF-1 and IGFBP-3 and IGF-1/IGFBP-3 molar ratio on mammographic density stratified by menopausal status. PATIENTS AND METHODS: A group of 341 Italian women were interviewed to collect the following data: family history of breast cancer, reproductive and menstrual factors, breast biopsies, previous administration of hormonal contraceptive therapy, hormone replacement therapy (HRT) in menopause and lifestyle information. A blood sample was drawn for determination of IGF-1, IGFBP-3 levels. IGF-1/ IGFBP-3 molar ratio was then calculated. On the basis of recent mammograms the women were divided into two groups: dense breast (DB) and non-dense breast (NDB). Student's t-test was employed to assess the association between breast density and plasma level of IGF-1, IGFBP-3 and molar ratio. To assess if this relationship was similar in subgroups of pre- and postmenopausal women, the study population was stratified by menopausal status and Student's t-test was performed. Finally, multivariate analysis was employed to evaluate if there were confounding factors that might influence the relationship between growth factors and breast density. RESULTS: The analysis of the relationship between mammographic density and plasma level of IGF-1, IGFBP-3 and IGF-1/ IGFBP-3 molar ratio showed that IGF-1 levels and molar ratio varied in the two groups resulting in higher mean values in the DB group (IGF-1: 109.6 versus 96.6 ng/ml; p= 0.001 and molar ratio 29.4 versus 25.5 ng/ml; p= 0.001) whereas IGFBP-3 showed similar values in both groups (DB and NDB). Analysis of plasma level of IGF-1, IGFBP-3 and IGF-1/IGFBP-3 molar ratio compared to breast density after stratification of the study population by menopausal status (premenopausal and postmenopausal) showed that there was no association between the plasma of growth factors and breast density, neither in premenopausal nor in postmenopausal patients. Multivariate analysis showed that only nulliparity, premenopausal status and body mass index (BMI) are determinants of breast density. CONCLUSIONS: Our study provides a strong evidence of a crude association between breast density and plasma levels of IGF-1 and molar ratio. On the basis of our results, it is reasonable to assume that the role of IGF-1 and molar ratio in the pathogenesis of breast cancer might be mediated through mammographic density. IGF-1 and molar ratio might thus increase the risk of cancer by increasing mammographic density.

[Nephrotic proteinuria with type 2 diabetes mellitus and autoimmune thyroiditis]. / Proteinuria nefrosica in paziente con diabete mellito di tipo II e tiroidite autoimmune.

This study reports on a 67-year-old man, suffering from type 2 diabetes mellitus for 11 years along with arterial hypertension and autoimmune thyroiditis, in whom nephrotic proteinuria was detected together with a mild reduction in GFR. No autoantibodies or monoclonal proteins were detected in blood and urine. Renal biopsy material examined by light microscopy, immunofluorescence and electron microscopy showed AL amyloidosis. This case underlines the role of renal biopsy in patients with type 2 diabetes mellitus, in whom renal diseases other than diabetic nephropathy may occur frequently.

Diagnosis, treatment, and response assessment in solitary plasmacytoma: updated recommendations from a European Expert Panel.

Solitary plasmacytoma is an infrequent form of plasma cell dyscrasia that presents as a single mass of monoclonal plasma cells, located either extramedullary or intraosseous. In some patients, a bone marrow aspiration can detect a low monoclonal plasma cell infiltration which indicates a high risk of early progression to an overt myeloma disease. Before treatment initiation, whole body positron emission tomography-computed tomography or magnetic resonance imaging should be performed to exclude the presence of additional malignant lesions. For decades, treatment has been based on high-dose radiation, but studies exploring the potential benefit of systemic therapies for high-risk patients are urgently needed. In this review, a panel of expert European hematologists updates the recommendations on the diagnosis and management of patients with solitary plasmacytoma.

International uniform response criteria for multiple myeloma.

New uniform response criteria are required to adequately assess clinical outcomes in myeloma. The European Group for Blood and Bone Marrow Transplant/International Bone Marrow Transplant Registry criteria have been expanded, clarified and updated to provide a new comprehensive evaluation system. Categories for stringent complete response and very good partial response are added. The serum free light-chain assay is included to allow evaluation of patients with oligo-secretory disease. Inconsistencies in prior criteria are clarified making confirmation of response and disease progression easier to perform. Emphasis is placed upon time to event and duration of response as critical end points. The requirements necessary to use overall survival duration as the ultimate end point are discussed. It is anticipated that the International Response Criteria for multiple myeloma will be widely used in future clinical trials of myeloma.

[Multimodal treatment complications of post-cholecystectomy intrahepatic lithiasis. Case report]. / Complicanze del trattamento multimodale della calcolosi intraepatica nel colecistectomizzato. Case report.

The Authors presents a case of post-cholecystectomy intrahepatic lithiasis. They focalized their attention to the complications of multimodal treatment.

Genome-wide association study of immunoglobulin light chain amyloidosis in three patient cohorts: comparison with myeloma.

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10-5 with homogeneity of results from the 3 sample sets; some of these were previously documented to influence MM risk, including the SNP at the IRF4 binding site. In AL amyloidosis, rs9344 at the splice site of cyclin D1, promoting translocation (11;14), reached the highest significance, P=7.80 × 10-11; the SNP was only marginally significant in MM. SNP rs79419269 close to gene SMARCD3 involved in chromatin remodeling was also significant (P=5.2 × 10-8). These data provide evidence for common genetic susceptibility to AL amyloidosis and MM. Cyclin D1 is a more prominent driver in AL amyloidosis than in MM, but the links to aggregation of light chains need to be demonstrated.

Rationale, application and clinical qualification for NT-proBNP as a surrogate end point in pivotal clinical trials in patients with AL amyloidosis.

Amyloid light-chain (LC) amyloidosis (AL amyloidosis) is a rare and fatal disease for which there are no approved therapies. In patients with AL amyloidosis, LC aggregates progressively accumulate in organs, resulting in organ failure that is particularly lethal when the heart is involved. A significant obstacle in the development of treatments for patients with AL amyloidosis, as well as for those with any disease that is rare, severe and heterogeneous, has been satisfying traditional clinical trial end points (for example, overall survival or progression-free survival). It is for this reason that many organizations, including the United States Food and Drug Administration through its Safety and Innovation Act Accelerated Approval pathway, have recognized the need for biomarkers as surrogate end points. The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. Underlying this consensus is the demonstration that NT-proBNP is an indicator of cardiac response in all interventional studies in which it has been assessed, despite differences in patient population, treatment type and treatment schedule. Furthermore, NT-proBNP expression is directly modulated by amyloidogenic LC-elicited signal transduction pathways in cardiomyocytes. The use of NT-proBNP will greatly facilitate the development of targeted therapies for AL amyloidosis. Here, we review the data supporting the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC and universally accepted by AL amyloidosis specialists, as a surrogate end point for survival.

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.

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.

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.

Quality of life in rheumatoid arthritis: impact of disability and lifetime depressive spectrum symptomatology.

OBJECTIVE: The aim of this study was to investigate the impact of disability and lifetime subthreshold depressive symptoms on Health-Related Quality of Life (HRQoL) among patients with rheumatoid arthritis (RA). METHODS: Ninety-two subjects with a diagnosis of RA according to the American College of Rheumatology (ACR) criteria were recruited at the Department of Rheumatology of the University Hospital, Pisa, Italy. Participants who met DSM-IV-TR diagnostic criteria for current or previous Axis I disorders were excluded. Assessments of functional status and disability was conducted using both the ACR classification and the Stanford Health Assessment Questionnaire (HAQ). Health-related Quality of Life was assessed using the Medical Outcomes Study Short Form 36 health survey questionnaire (MOS-SF36) and lifetime depressive spectrum symptomatology using the Mood Spectrum Questionnaire, Self-Report version (MOODS-SR). RESULTS: Comparison with MOS-SF36 Italian normative values indicated that RA patients were significantly impaired on mental and physical HRQoL areas. Correlations between MOODS-SR depressive scores and ACR severity (Spearman rho = 0.15, p = 0.07) and HAQ score (Spearman rho = 0.20, p = 0.05) were modest in absolute value and borderline significant. Lifetime mood depressive spectrum was related with impaired HRQoL levels, both in physical (except for bodily pain) and mental (except for social functioning) domains. Associations of mood depressive spectrum and general health, vitality, role emotional and mental health continued to be significant after controlling for functional status, duration of illness, age and gender. CONCLUSIONS: Because lifetime mood depressive symptoms significantly contribute to impairment in HRQoL in RA patients without a past psychiatric history, even after controlling for functional status, duration of illness and demographic characteristics, these symptoms should be assessed for an accurate clinical evaluation and appropriate clinical management of RA patients.

Biological features of the clone involved in primary amyloidosis (AL).

Primary light chain-associated amyloidosis (AL) is a plasma cell dyscrasia that causes morbidity via systemic tissue deposition of monoclonal light chains in the form of fibrils (amyloid). It is the most common form of systemic amyloidosis in Western countries and is rapidly fatal. Knowledge of the pathobiology of the underlying B cell clone is of primary importance for the design and optimization of therapeutic strategies.

Effects of a new aminodiphosphonate (aminohydroxybutylidene diphosphonate) in patients with osteolytic lesions from metastases and myelomatosis. Comparison with dichloromethylene diphosphonate.

We have compared in an open trial the clinical and biochemical effects of a new aminodiphosphonate, aminohydroxybutylidene diphosphonate, with those of dichloromethylene diphosphonate, which has been proved effective. The patients presented extensive and symptomatic bone involvement from multiple myeloma, breast cancer, and other metastatic tumors. The treatment consisted of aminohydroxybutylidene diphosphonate, 2.5 mg/d intravenously for five days, or dichloromethylene diphosphonate, 300 mg/d intravenously for seven days, followed by 100 mg/d intramuscularly for ten days. Twelve patients treated with aminohydroxybutylidene diphosphonate and 16 patients treated with dichloromethylene diphosphonate were assessable and were followed up for one to six months. Therapy with aminohydroxybutylidene diphosphonate showed a quicker action in reducing bone pain and reduced significantly more the serum calcium level than did therapy with dichloromethylene diphosphonate. Aminohydroxybutylidene diphosphonate therapy also affected urinary calcium levels and hydroxyproline excretion more markedly than did dichloromethylene diphosphonate, although the differences are not statistically significant. However, the biochemical indexes rebounded more quickly in patients treated with aminohydroxybutylidene diphosphonate, indicating that the loading amount (only 12.5 mg) used in this preliminary study is insufficient to sustain a prolonged effect. The effectiveness and lack of side effects render aminohydroxybutylidene diphosphonate an attractive treatment for malignant bone resorption.

[Primary Sjogren's syndrome: clinical and serological feature of a single centre]. / Sindrome di Sjogren primitiva: descrizione clinica e sierologica di una casistica monocentrica.

OBJECTIVE: To describe clinical and serological features of a large series of patients affected by primary Sjogren's syndrome (pSS), assessing the evolution of the disease in a long-term follow-up study. METHODS: Clinical and laboratory data of 250 patients with pSS attending our Unit for a mean follow-up period of 140 months were retrospectively collected and analysed. In all the cases the diagnosis was made according with the recent international criteria. RESULTS: Glandular involvement was almost universally present, typically as the first manifestation of the disease and a slow progression of the salivary and lachrymal dysfunction was seen during the observation period. Extraglandular involvement was mild, quite rare and delayed. The respective percentages for muscle-skeletal disease, urogenital, haematological, skin, pulmonary, gastrointestinal, neurological and renal involvement were 60%, 40%, 24%, 20%, 11%, 7%, 8% and 3%. Only 6 patients developed a lymphoma. The serological pattern of the majority of patients remained constant throughout the follow-up period. CONCLUSIONS: pSS is often a benign condition. Since some patients may develop lymphoid malignancies, clinical follow-up is recommended.

The second riboflavin-binding myeloma IgG lambdaDOT. I. Biochemical and functional characterization.

A human monoclonal immunoglobulin, IgGDOT, with flavin-binding capacity has been obtained from an elderly woman with multiple myeloma who developed yellow skin and yellow hair. The case presented a remarkable similarity with that previously reported by Farhangi and Osserman [N. Engl. J. Med. 294, 177-183 (1976)]. Purified IgGDOT was bright yellow and the ligand was identified as riboflavin and its oxidation products by thin layer chromatography, proton nuclear magnetic resonance and mass spectroscopy. Competitive binding studies with different haptens demonstrated highest affinity for riboflavin, followed by flavin mononucleotide and flavin adenine dinucleotide; no significant binding was detected for several other non-flavin compounds tested. The hapten was associated with the protein in vivo, as well as with the purified antibody. Removal of the already bound riboflavin from the combining site was associated with irreversible denaturation of the monoclonal protein. By the fluorescence quenching technique it was determined that there were 0.68 available combining sites for riboflavin molecule in IgGDOT with a binding constant of 8.5 x 10(8)/M, while FabDOT presented 0.27 available combining sites with a binding constant of 5.1 x 10(8)/M. The fact that 1.2 and 0.81 mol riboflavin/mol protein were already bound to IgGDOT and FabDOT, respectively, is consistent with the usual hapten/antibody stoichiometry. The heavy chain subclass of IgGDOT was identified as gamma 2, as in the previously reported case of riboflavin-binding protein IgGGAR. However, the lambda chain subclass was different and no idiotype cross-reactivity was found.

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.