Philip N. Hawkins

NALP3 Forms an IL-1β-Processing Inflammasome with Increased Activity in Muckle-Wells Autoinflammatory Disorder

Mutations within the NALP3/cryopyrin/CIAS1 gene are responsible for three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. The NALP3 protein is homologous to NALP1, which is a component of the inflammasome, a molecular platform that activates the proinflammatory caspases-1 and -5. NALP3 (and other members of the NALP family) lacks the C-terminal, CARD-containing sequence of NALP1, and its role in caspase activation is unclear. Here, we report that NALP2 and NALP3 associate with ASC, the CARD-containing protein Cardinal, and caspase-1 (but not caspase-5), thereby forming an inflammasome with high proIL-1beta-processing activity. Macrophages from Muckle-Wells patients spontaneously secrete active IL-1beta. Increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with NALP3-dependent autoinflammatory disorders.

Cardiovascular Magnetic Resonance in Cardiac Amyloidosis

Background—Cardiac amyloidosis can be diagnostically challenging. Cardiovascular magnetic resonance (CMR) can assess abnormal myocardial interstitium. Methods and Results—Late gadolinium enhancement CMR was performed in 30 patients with cardiac amyloidosis. In 22 of these, myocardial gadolinium kinetics with T1 mapping was compared with that in 16 hypertensive controls. One patient had CMR and autopsy only. Subendocardial T1 in amyloid patients was shorter than in controls (at 4 minutes: 427±73 versus 579±75 ms; P<0.01), was shorter than subepicardium T1 for the first 8 minutes (P≤0.01), and was correlated with markers of increased myocardial amyloid load, as follows: left ventricular (LV) mass (r=−0.51, P=0.013); wall thickness (r=−0.54 to −0.63, P<0.04); interatrial septal thickness (r=−0.52, P=0.001); and diastolic function (r=−0.42, P=0.025). Global subendocardial late gadolinium enhancement was found in 20 amyloid patients (69%); these patients had greater LV mass (126±30 versus 93±25 g/m2; P=0.009) than unenhanced patients. Histological quantification showed substantial interstitial expansion with amyloid (30.5%) but only minor fibrosis (1.3%). Amyloid was dominantly subendocardial (42%) compared with midwall (29%) and subepicardium (18%). There was 97% concordance in diagnosis of cardiac amyloid by combining the presence of late gadolinium enhancement and an optimized T1 threshold (191 ms at 4 minutes) between myocardium and blood. Conclusions—In cardiac amyloidosis, CMR shows a characteristic pattern of global subendocardial late enhancement coupled with abnormal myocardial and blood-pool gadolinium kinetics. The findings agree with the transmural histological distribution of amyloid protein and the cardiac amyloid load and may prove to have value in diagnosis and treatment follow-up.

Use of Canakinumab in the Cryopyrin-Associated Periodic Syndrome

BACKGROUND The cryopyrin-associated periodic syndrome (CAPS) is a rare inherited inflammatory disease associated with overproduction of interleukin-1. Canakinumab is a human anti-interleukin-1beta monoclonal antibody. METHODS We performed a three-part, 48-week, double-blind, placebo-controlled, randomized withdrawal study of canakinumab in patients with CAPS. In part 1, 35 patients received 150 mg of canakinumab subcutaneously. Those with a complete response to treatment entered part 2 and were randomly assigned to receive either 150 mg of canakinumab or placebo every 8 weeks for up to 24 weeks. After the completion of part 2 or at the time of relapse, whichever occurred first, patients proceeded to part 3 and received at least two more doses of canakinumab. We evaluated therapeutic responses using disease-activity scores and analysis of levels of C-reactive protein (CRP) and serum amyloid A protein (SAA). RESULTS In part 1 of the study, 34 of the 35 patients (97%) had a complete response to canakinumab. Of these patients, 31 entered part 2, and all 15 patients receiving canakinumab remained in remission. Disease flares occurred in 13 of the 16 patients (81%) receiving placebo (P<0.001). At the end of part 2, median CRP and SAA values were normal (<10 mg per liter for both measures) in patients receiving canakinumab but were elevated in those receiving placebo (P<0.001 and P=0.002, respectively). Of the 31 patients, 28 (90%) completed part 3 in remission. In part 2, the incidence of suspected infections was greater in the canakinumab group than in the placebo group (P=0.03). Two serious adverse events occurred during treatment with canakinumab: one case of urosepsis and an episode of vertigo. CONCLUSIONS Treatment with subcutaneous canakinumab once every 8 weeks was associated with a rapid remission of symptoms in most patients with CAPS. (ClinicalTrials.gov number, NCT00465985.)

Metabolic and scintigraphic studies of radioiodinated human C-reactive protein in health and disease.

Plasma and whole-body turnover studies of human C-reactive protein (CRP), isolated from a single normal healthy donor and labeled with 125I, were undertaken in 8 healthy control subjects and 35 hospitalized patients including cases of rheumatoid arthritis, systemic lupus erythematosus, infections, and neoplasia. Plasma clearance of 125I-CRP closely approximated to a monoexponential function and was similar in the control and all patient groups. There was no evidence for accelerated clearance or catabolism of CRP in any of the diseases studied. The 19-h half-life was more rapid than that of most human plasma proteins studied previously, and the fractional catabolic rate was independent of the plasma CRP concentration. The synthesis rate of CRP is thus the only significant determinant of its plasma level, confirming the validity of serum CRP measurement as an objective index of disease activity in disorders associated with an acute-phase response. Approximately 90% of injected radioactivity was recovered in the urine after 7 d, and scintigraphic imaging studies with 123I-labeled CRP in 10 patients with different focal pathology showed no significant localization of tracer. The functions of CRP are thus likely to be effected predominantly in the fluid phase rather than by major deposition at sites of tissue damage or inflammation.

Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

BACKGROUND Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis.

The normal plasma protein serum amyloid P component (SAP) binds to fibrils in all types of amyloid deposits, and contributes to the pathogenesis of amyloidosis. In order to intervene in this process we have developed a drug, R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, that is a competitive inhibitor of SAP binding to amyloid fibrils. This palindromic compound also crosslinks and dimerizes SAP molecules, leading to their very rapid clearance by the liver, and thus produces a marked depletion of circulating human SAP. This mechanism of drug action potently removes SAP from human amyloid deposits in the tissues and may provide a new therapeutic approach to both systemic amyloidosis and diseases associated with local amyloid, including Alzheimers disease and type 2 diabetes.

Amyloid load and clinical outcome in AA amyloidosis in relation to circulating concentration of serum amyloid A protein.

BACKGROUND Reactive systemic (AA, secondary) amyloidosis occurs in chronic inflammatory diseases, and most patients present with nephropathy. The amyloid fibrils are derived from the circulating acute-phase reactant serum amyloid A protein (SAA), but the relation between production of fibril precursor protein, amyloid load, and clinical outcome in AA and other types of amyloidosis is unclear. METHODS We studied amyloidotic organ function and survival prospectively for 12-117 months in 80 patients with systemic AA amyloidosis in whom serum SAA concentration was measured monthly and visceral amyloid deposits were assessed annually by serum amyloid P component scintigraphy. Underlying inflammatory diseases were treated as vigorously as possible. FINDINGS Amyloid deposits regressed in 25 of 42 patients whose median SAA values were within the reference range (<10 mg/L) throughout follow-up, and amyloidotic organ function stabilised or improved in 39 of these cases. Outcome varied substantially among patients whose median SAA concentration exceeded 10 mg/L, but amyloid load increased and organ function deteriorated in most of those whose SAA was persistently above 50 mg/L. Estimated survival at 10 years was 90% in patients whose median SAA was under 10 mg/L, and 40% among those whose median SAA exceeded this value (p=0.0009). INTERPRETATION Although isolated amyloid fibrils are stable in vitro, AA amyloid deposits exist in a state of dynamic turnover, and outcome is favourable in AA amyloidosis when the SAA concentration is maintained below 10 mg/L. The potential for amyloid to regress and for the function of amyloidotic organs to recover support therapeutic strategies to decrease the supply of amyloid fibril precursor proteins in amyloidosis generally.

Interleukin-1–Receptor Antagonist in the Muckle–Wells Syndrome

To the Editor: Studies of hereditary inflammatory disorders have identified novel genes and pathways that may be involved in inflammation and apoptosis generally. Mutations in one such gene, variou...

Evaluation of Systemic Amyloidosis by Scintigraphy with 123I-Labeled Serum Amyloid P Component

BACKGROUND In systemic amyloidosis the distribution and progression of disease have been difficult to monitor, because they can be demonstrated only by biopsy. Serum amyloid P component (SAP) is a normal circulating plasma protein that is deposited on amyloid fibrils because of its specific binding affinity for them. We investigated whether labeled SAP could be used to locate amyloid deposits. METHODS Purified human SAP labeled with iodine-123 was given intravenously to 50 patients with biopsy-proved systemic amyloidosis--25 with the AL (primary) type and 25 with the AA (secondary) type--and to 26 control patients with disease and 10 healthy subjects. Whole-body images and regional views were obtained after 24 hours and read in a blinded fashion. RESULTS In the patients with amyloidosis the 123I-SAP was localized rapidly and specifically in amyloid deposits. The scintigraphic images obtained were characteristic and appeared to identify the extent of amyloid deposition in all 50 patients. There was no uptake of the 123I-SAP by the control patients and the healthy subjects. In all patients with AA amyloidosis the spleen was affected, whereas the scans showed uptake in the heart, skin, carpal region, and bone marrow only in patients with the AL type. Positive images were seen in six patients in whom biopsies had been negative or unsuccessful; in all six, amyloid was subsequently found on biopsy or at autopsy. Progressive amyloid deposition was observed in 9 of 11 patients studied serially. CONCLUSIONS Scintigraphy after the injection of 123I-SAP can be used for diagnosing, locating, and monitoring the extent of systemic amyloidosis.

Outcome in systemic AL amyloidosis in relation to changes in concentration of circulating free immunoglobulin light chains following chemotherapy.

Summary. Monoclonal immunoglobulin light chains are deposited as amyloid fibrils in systemic AL (primary) amyloidosis, but the underlying plasma cell dyscrasias are often difficult to detect or unquantifiable. The relationships between circulating monoclonal light chains, amyloid load and clinical outcome, and the relative efficacies of chemotherapy regimens aimed at suppressing monoclonal immunoglobulin production, have not been determined. Circulating free immunoglobulin light chain (FLC) concentration was measured with a sensitive nephelometric immunoassay in 262 patients with AL amyloidosis, and followed serially in 137 patients who received either high‐dose chemotherapy or one of two intermediate‐dose cytotoxic regimens. Amyloid load was quantified by serum amyloid P component scintigraphy. A monoclonal excess of FLC was identified at diagnosis in 98% of patients. Among 86 patients whose abnormal FLC concentration fell by more than 50% following chemotherapy, 5‐year survival was 88% compared with only 39% among those whose FLC did not fall by half (P < 0·0001). Amyloid deposits regressed in 58 patients. The magnitude and duration of the FLC responses to intermediate‐ and high‐dose chemotherapy regimens were similar. The FLC assay enabled the circulating fibril precursor protein in AL amyloidosis to be quantified and monitored in most patients. Reduction of the amyloidogenic FLC by more than 50% was associated with substantial survival benefit, regardless of the type of chemotherapy used. Clinical improvement following chemotherapy in AL amyloidosis is delayed, but treatment strategies can be guided by their early effect on serum FLC concentration.