Bouke Hazenberg

Misfolded proteins activate Factor XII in humans, leading to kallikrein formation without initiating coagulation

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.

Nonbiopsy Diagnosis of Cardiac Transthyretin Amyloidosis

Background— Cardiac transthyretin (ATTR) amyloidosis is a progressive and fatal cardiomyopathy for which several promising therapies are in development. The diagnosis is frequently delayed or missed because of the limited specificity of echocardiography and the traditional requirement for histological confirmation. It has long been recognized that technetium-labeled bone scintigraphy tracers can localize to myocardial amyloid deposits, and use of this imaging modality for the diagnosis of cardiac ATTR amyloidosis has lately been revisited. We conducted a multicenter study to ascertain the diagnostic value of bone scintigraphy in this disease. Methods and Results— Results of bone scintigraphy and biochemical investigations were analyzed from 1217 patients with suspected cardiac amyloidosis referred for evaluation in specialist centers. Of 857 patients with histologically proven amyloid (374 with endomyocardial biopsies) and 360 patients subsequently confirmed to have nonamyloid cardiomyopathies, myocardial radiotracer uptake on bone scintigraphy was >99% sensitive and 86% specific for cardiac ATTR amyloid, with false positives almost exclusively from uptake in patients with cardiac AL amyloidosis. Importantly, the combined findings of grade 2 or 3 myocardial radiotracer uptake on bone scintigraphy and the absence of a monoclonal protein in serum or urine had a specificity and positive predictive value for cardiac ATTR amyloidosis of 100% (positive predictive value confidence interval, 98.0–100). Conclusions— Bone scintigraphy enables the diagnosis of cardiac ATTR amyloidosis to be made reliably without the need for histology in patients who do not have a monoclonal gammopathy. We propose noninvasive diagnostic criteria for cardiac ATTR amyloidosis that are applicable to the majority of patients with this disease.

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.

Improvement of Lipid Profile Is Accompanied by Atheroprotective Alterations in High-Density Lipoprotein Composition Upon Tumor Necrosis Factor Blockade A Prospective Cohort Study in Ankylosing Spondylitis

OBJECTIVE Cardiovascular mortality is increased in ankylosing spondylitis (AS), and inflammation plays an important role. Inflammation deteriorates the lipid profile and alters high-density lipoprotein cholesterol (HDL-c) composition, reflected by increased concentrations of serum amyloid A (SAA) within the particle. Anti-tumor necrosis factor (anti-TNF) treatment may improve these parameters. We therefore undertook the present study to investigate the effects of etanercept on lipid profile and HDL composition in AS. METHODS In 92 AS patients, lipid levels and their association with the inflammation markers C-reactive protein (CRP), erythrocyte sedimentation rate, and SAA were evaluated serially during 3 months of etanercept treatment. HDL composition and its relationship to inflammation markers was determined in a subgroup of patients, using surface-enhanced laser desorption/ionization time-of-flight analysis. RESULTS With anti-TNF treatment, levels of all parameters of inflammation decreased significantly, whereas total cholesterol, HDL-c, and apolipoprotein A-I (Apo A-I) levels increased significantly. This resulted in a better total cholesterol:HDL-c ratio (from 3.9 to 3.7) (although the difference was not statistically significant), and an improved Apo B:Apo A-I ratio, which decreased by 7.5% over time (P=0.008). In general, increases in levels of all lipid parameters were associated with reductions in inflammatory activity. In addition, SAA was present at high levels within HDL particles from AS patients with increased CRP levels and disappeared during treatment, in parallel with declining plasma levels of SAA. CONCLUSION Our results show for the first time that during anti-TNF therapy for AS, along with favorable changes in the lipid profile, HDL composition is actually altered whereby SAA disappears from the HDL particle, increasing its atheroprotective ability. These findings demonstrate the importance of understanding the role of functional characteristics of HDL-c in cardiovascular diseases related to chronic inflammatory conditions.

Erythrocyte Sedimentation Rate, C-Reactive Protein Level, and Serum Amyloid A Protein for Patient Selection and Monitoring of Anti-Tumor Necrosis Factor Treatment in Ankylosing Spondylitis

OBJECTIVE To study the usefulness of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum amyloid A (SAA) for response prediction and monitoring of anti-tumor necrosis factor (anti-TNF) treatment in ankylosing spondylitis (AS) patients. METHODS Patients were included consecutively before starting etanercept or infliximab treatment. ASsessment in Ankylosing Spondylitis (ASAS) response, defined as a 50% improvement or an absolute improvement of 2 points of the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI; 0-10 scale), was assessed at 3 months. Inflammatory markers and the BASDAI were collected at baseline and 1 and 3 months. Longitudinal data analysis was performed to compare associations between inflammatory markers and the BASDAI over time by calculating standardized betas. Predictive values of baseline levels of inflammatory markers for ASAS response were calculated. RESULTS In total, 155 patients were included, of whom, after 3 months of treatment, 70% in the etanercept cohort and 71% in the infliximab cohort responded. All markers, notably SAA, decreased significantly (P < 0.0001). Standardized betas were 0.49 for ESR, 0.43 for CRP, and 0.39 for SAA. Normal baseline levels of CRP and SAA were significantly associated with nonresponse. A combination of elevated CRP and SAA levels at baseline revealed the highest predictive value (81%) for ASAS response. CONCLUSION ESR, CRP, and SAA were significantly associated with the BASDAI over 3 months, and the association with ESR was the strongest. Elevated baseline CRP and SAA levels revealed the highest predictive value for response. Together, this study demonstrates that inflammatory markers, and notably CRP and SAA, may facilitate patient selection and monitoring of efficacy of anti-TNF treatment in AS, and could be added to response criteria.

Where has secondary amyloid gone

There is a growing clinical impression that the incidence of amyloid secondary to rheumatoid disease is declining. Recent studies from Finland strongly support this impression.1-3 The prevalence of amyloidosis in patients with rheumatoid arthritis who died in 1989 (about 6%)1 was lower than in earlier studies.4 In a recent cohort of patients with early rheumatoid arthritis followed up for 8–14 years no one died from amyloidosis.2 In a hospital for rheumatic diseases the annual number of biopsies positive for amyloid decreased from more than 60 in 1987 to fewer than 10 recently.3 The question arises whether this is a true decline of the incidence or a more concealed clinical presentation of this type of amyloidosis. Secondary amyloidosis is nowadays called systemic AA amyloidosis. It is associated with chronic inflammation and results from systemic deposition of the acute phase reactant serum amyloid A protein (SAA) in a fibrillar structure. SAA behaves similarly to C reactive protein, being hardly detectable in normal situations and increasing quickly and dramatically during inflammation.5 In selected populations of patients with rheumatoid arthritis 5–20% of patients with longstanding disease will develop AA amyloidosis, depending upon the severity and duration of the arthritis in the population investigated.6 7 Although longstanding inflammation is necessary for the development of AA amyloidosis, it is not sufficient. Most people with a longstanding increase in SAA levels will never develop AA amyloidosis. In Europe AA amyloidosis is seen more often than in North America. Food, lifestyle, differences in drug treatment, other concurrent diseases, and genetic factors might be responsible for this difference. Genetic predisposition may be conditional for amyloid formation. One possible factor is homozygosity for SAA subtypes.8 9 Genetic research of diseases connected to the development of AA amyloidosis, such as familial Mediterranean fever, …

Nuclear imaging in cardiac amyloidosis

Amyloidosis is a disease characterized by depositions of amyloid in organs and tissues. It can be localized (in just one organ) or systemic. Cardiac amyloidosis is a debilitating disease and can lead to arrhythmias, deterioration of heart function and even sudden death. We reviewed PubMed/Medline, without time constraints, on the different nuclear imaging modalities that are used to visualize myocardial amyloid involvement. Several SPECT tracers have been used for this purpose. The results with these tracers in the evaluation of myocardial amyloidosis and their mechanisms of action are described. Most clinical evidence was found for the use of 123I-MIBG. Myocardial defects in MIBG activity seem to correlate well with impaired cardiac sympathetic nerve endings due to amyloid deposits. 123I-MIBG is an attractive option for objective evaluation of cardiac sympathetic level and may play an important role in the indirect measurement of the effect of amyloid myocardial infiltration. Other, less sensitive, options are 99mTc-aprotinin for imaging amyloid deposits and perhaps 99mTc-labelled phosphate derivatives, especially in the differential diagnosis of the aetiology of cardiac amyloidosis. PET tracers, despite the advantage of absolute quantification and higher resolution, are not yet well evaluated for the study of cardiac amyloidosis. Because of these advantages, there is still the need for further research in this field.

Clinical and therapeutic aspects of AA amyloidosis

Approach to the management of AA amyloidosis complicating RA. (A) In case of proteinuria or loss of renal function a rectal biopsy or a subcutaneous fat biopsy is a suitable screening method for the detection of amyloidosis. If in any doubt, try to ascertain the diagnosis by renal biopsy. Adequate staining with alkaline Congo red and preferably immunohistochemical staining with anti-AA antibodies should be performed. Beware of renal pathology other than amyloidosis even in the presence of a positive rectal biopsy. (B) A vigorous attempt to control disease activity of the RA should be made in order to eliminate the production of SAA, an acute phase protein. The response to treatment should be monitored by serial measurements of CRP and preferably SAA

Bone scintigraphy with (99m)technetium-hydroxymethylene diphosphonate allows early diagnosis of cardiac involvement in patients with transthyretin-derived systemic amyloidosis

Abstract Objective: To assess the usefulness of bone scintigraphy with 99mTechnetium-hydroxymethylene diphosphonate (99mTc-HDP) for the detection of cardiac involvement in a group of patients with ATTR amyloidosis in different phases of disease, to relate the findings to echocardiography, ECG and cardiac biomarkers, and to evaluate different bone scintigraphic techniques and calculation methods for quantification of the cardiac uptake and for correlation with echocardiographic features and cardiac biomarkers. Methods: Forty-one patients underwent clinical examinations, echocardiography, ECG, measurement of cardiac biomarkers and bone scintigraphy (planar imaging and SPECT-CT) and were subsequently subdivided into three groups: (1) carriers of an amyloidogenic TTR mutation, n = 11, (2) proven ATTR amyloidosis without echocardiographically-defined (mean wall thickness >12 mm) cardiac amyloidosis (AC), n = 19, and (3) ATTR amyloidosis with echocardiographically-defined cardiac amyloidosis, n = 11. Planar and SPECT-CT images were analyzed visually according to a routine scoring system (grade 0–3) and semi-quantitatively by heart-to-whole body (H/WB) and heart-to-skull (H/S) ratio on planar images and by a left ventricle-blood pool ratio on SPECT-CT images. Results: All patients with ATTR and echocardiographically-defined AC and none of the carriers showed high cardiac uptake on bone scintigraphy. Furthermore, 8 out of 19 patients with ATTR without echocardiographically-defined AC showed high cardiac uptake. Highest correlations were found between H/S ratio on planar bone scintigraphy with troponin T (r = 0.76, p < 0.0001) and H/WB ratio with left ventricular mass index (r = 0.73, p < 0.0001). Conclusions: Bone scintigraphy with 99mTc-HDP may detect cardiac involvement in patients with ATTR amyloidosis prior to echocardiographic evidence of cardiac involvement. Cardiac uptake on bone scintigraphy correlates with severity of cardiac involvement using echocardiography, ECG and cardiac biomarkers. Visual grading and calculation of H/S ratio on planar imaging are the preferred methods to assess cardiac uptake.

Amyloidosis: A Clinical Overview

This issue contains two contributions on amyloidosis (p. 545 and p. 548). A previous communication was published last year (Wahi, 1950). In these communications references have been made to other cases described recently. Apparently a rare disease is raising its head. In this morbid state a hyalin-like material is deposited between cells, in the formed elements. It selects specially fine connective tissue fibrils in relation to blood vessels (Cappell, 1951). The material, in spite of its name, is apparently protein in nature, probably a combination of albumin and chondroitin-sulphuric acid, derived from a long-continued destruction of organs rich in elastic connective tissue (Hyman, 1946). Variability in staining suggests a series of varying but closely-related proteins (Anderson, 1948). Experimentally chondroitin-sulphuric acid does not produce amyloidosis. Some investigators hold the reticuloendothelial system responsible for the formation of the material. Others look upon the material as a kind of antigen-antibody precipitate. Some conditions in which it is found are definitely associated with hyperglobulinaemia, such as hyper-immunization of horses: This lends support to the concept of antigen-antibody precipitate. Excessive feeding of mice with cheese and other proteins also is known to give rise to the material. Repeated injections of bacteria and nutrose (neutral casein sodium) into animals are also known to cause amyloidosis : This brings one back to cheese and (hyper-) immunization. Metabolic and nutritional disturbances other than overfeeding should also be kept in mind. The tissues infiltrated with the deposit stain characteristically deeply with iodine (Lugol solution). Congo red is used in selective staining. It is also used as a test of the disease, because of its fixation by the affected living tissue. 15 cc. of a 0.75 per cent solutionAmyloidosis is the name for protein-folding diseases characterized by extracellular deposition of a specific soluble precursor protein that aggregates in the form of insoluble fibrils. The classification of amyloidosis is based on the chemical characterization of the precursor protein. Deposition of amyloid is localized or systemic. The 4 main types of systemic amyloidosis are AL, AA, ATTR, and Aβ2M type. A schematic approach is proposed for the clinical management of systemic amyloidosis. The importance of typing amyloid with confidence, the usefulness of imaging techniques, the principles of treatment, and the need for well-planned treatment monitoring during follow-up are discussed.