Bouke P. C. Hazenberg

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.

Sjögren's syndrome and localized nodular cutaneous amyloidosis: coincidence or a distinct clinical entity?

OBJECTIVEnTo report 8 patients with Sjögrens syndrome (SS) and localized nodular cutaneous amyloidosis and to examine serologic and immunohistologic findings that may link the 2 diseases.nnnMETHODSnThe databases for 3 amyloidosis centers were searched for patients with localized nodular cutaneous amyloidosis and SS. Eight patients with this combination were identified, and clinical, serologic, and histologic parameters were retrospectively evaluated.nnnRESULTSnAmong the 8 patients with a clinical diagnosis of SS, 6 fulfilled the American-European Consensus Group criteria for SS. All of the patients were women in whom SS had been diagnosed at a median age of 47 years (range 30-61 years) and amyloid had been diagnosed at a median age of 60 years (range 42-79 years). The presence of the immunoglobulin light chain type of amyloid (AL amyloid) was confirmed in 4 patients. In 3 of these 4 patients as well as 2 other patients, a light chain-restricted plasma cell population was observed near the amyloid deposits. Progression to systemic amyloidosis was not observed in any patient during a median followup of 3.5 years.nnnCONCLUSIONnSS should be considered in patients with cutaneous amyloidosis. The combination of cutaneous amyloidosis and SS appears to be a distinct disease entity reflecting a particular and benign part of the polymorphic spectrum of lymphoproliferative diseases related to SS.

123I-Labelled metaiodobenzylguanidine for the evaluation of cardiac sympathetic denervation in early stage amyloidosis

PurposeCardiac amyloidosis is a rare disorder, but it may lead to potentially life-threatening restrictive cardiomyopathy. Cardiac manifestations frequently occur in primary amyloidosis (AL) and familial amyloidosis (ATTR), but are uncommon in secondary amyloidosis (AA). Echocardiography is the method of choice for assessing cardiac amyloidosis. Amyloid deposits impair the function of sympathetic nerve endings. Disturbance of myocardial sympathetic innervations may play an important role in the remodelling process. 123I-MIBG can detect these innervation changes.MethodsPatients with biopsy-proven amyloidosis underwent general work-up, echocardiography and 123I-MIBG scintigraphy. Left ventricular internal dimensions and wall thickness were measured, and highly refractile cardiac echoes (sparkling) were analysed. Early (15xa0min) and late (4xa0h) heart-to-mediastinum ratio (HMR) and wash-out rate were determined after administration of MIBG.ResultsIncluded in the study were 61 patients (30 women and 31 men; mean age 62xa0years; 39 AL, 11 AA, 11 ATTR). Echocardiographic parameters were not significantly different between the groups. Sparkling was present in 72xa0% of ATTR patients, in 54xa0% of AL patients and in 45xa0% of AA patients. Mean late HMR in all patients was 2.3u2009±u20090.75, and the mean wash-out rate was 8.6u2009±u200914xa0% (the latter not significantly different between the patient groups). Late HMR was significantly lower in patients with echocardiographic signs of amyloidosis than in patients without (2.0u2009±u20090.70 versus 2.8u2009±u20090.58, pu2009<u20090.001). Wash-out rates were significantly higher in these patients (−3.3u2009±u20099.9xa0% vs. 17u2009±u200910xa0%, pu2009<u20090.001). In ATTR patients without echocardiographic signs of amyloidosis, HMR was lower than in patients with the other types (2.0u2009±u20090.59 vs. 2.9u2009±u20090.50, pu2009=u20090.007).ConclusionMIBG HMR is lower and wash-out rate is higher in patients with echocardiographic signs of amyloidosis. Also, 123I-MIBG scintigraphy can detect cardiac denervation in ATTR patients before signs of amyloidosis are evident on echocardiography.

SAA Versus CRP Serum Levels in Different Inflammatory Conditions, Studied by Elisa Using Polyclonal Anti-AA and Monoclonal Anti-SAA Antibodies

SAA was quantified by two newly developed sandwich ELISA’s. First antibody in both assays was a purified polyclonal rabbit anti-AA (PRαAA), bound to the microtitration plate. In one assay PRαAA was also used to detect the captured SAA, but in the second a monoclonal murine anti-SAA antibody (MMαSAA) was used for this detection. The results of both assays were comparable, so for routine purposes the more convenient and standardizable second assay was preferred. We used a highly purified apo-SAA, extracted from pooled acute-phase sera, as the ultimate standard for our assays. Purity was established by SDS-PAGE, immunoblotting and amino acid analysis. To obtain our standard we finally added an exact amount to normal negative serum. With this SAA-ELISA and a comparable one for CRP, simultaneous SAA and CRP levels were measured in CAH, PBC, hepatoma’s, NSTT, M.Crohn, CTD, RA and renal allograft rejection. The SAA/CRP ratio differed among these groups, ranging from a clear SAA preponderance in renal allograft rejection to a clear CRP preponderance in liver diseases.

Monoclonal Antibody Based ELISA for Human SAA

We developed an ELISA for human SAA in which two different monoclonal antibodies (MAb) were used (Reu.86.1 and Reu.86.5) that recognized different epitopes on SAA. These MAb’s were raised against purified SAA coupled to Helix pomatia haemocyanin (by glutaraldehyde). The MAb’s reacted with isolated AA-proteins and with SAA (all major isotypes), both isolated and after reconstitution in HDL3. The MAb’s could be used in ELISA, in immunohistochemistry, and in immuno-blotting after SDS-PAGE or IEF. In ELISA one of the MAb’s was coated to the microtiterplate (Reu.86.5), while the other (Reu.86.1) was coupled to HRPO. The internal standard in the ELISA consisted either of SAA isolated from a serum pool (150 patients with CRP >100 mg/1) reconstituted into normal serum or of a reference serum sample. The lower detection limit of the assay for SAA was 5 μg/1, allowing the use in biological fluids (serum, urine, synovial fluid) and in in-vitro systems (cultured human hepatocytes). In routine serology we found an intra-assay coefficient of variation (CV) and an inter-assay CV both >10% (N=15 res. N=17). In this assay no pretreatment of testsamples was necessary and there was no interference of rheumatoid factors. The basal reference values of healthy controls had a 95% upper limit of 2.6 mg/1. This SAA-ELISA uses well defined MAb’s for the detection of SAA, which permits standardization, inter-laboratory comparability and general availability.

Immunohistochemical Detection of Amyloid AA in Formaline-Fixed Paraffine-Embedded Rectal Biopsies with the Monoclonal Anti-Human Saa Antibody Reu.86.2

The present study addresses the reliability of a monoclonal anti-human SAA antibody (Reu. 86.2) to detect amyloid AA in formaline-fixed paraffine-embedded rectal biopsies. A total of 34 rectal biopsies obtained from amyloidotic patients from 1986 until februari 1990 were positive for amyloid in the Congo red (CR) stain. Pretreatment with KMnO4 eliminated CR staining in 30 specimens (AA-amyloid) whereas in 4 biopsies CR staining was resistent to KMnO4 (non-AA). A murine monoclonal antibody (Reu.86.2) was raised against apo-SAA coupled to Helix Pomatia Haemocyanin. After pretreatment with 0.1% protease routine paraffine sections were incubated with Reu.86.2 as the first layer and peroxidase-conjugated Rabbit anti-Mouse antibody as the second. All 30 KMnO4 sensitive specimens showed moderate or strong reactivity in immunostaining with the monoclonal antibody (in these biopsies the intensity of CR staining was moderate or strong in 18 and weak in 12). Twenty CR-negative controls and all 4 KMnO4-resistent biopsies were negative in immunohistochemistry with Reu.86.2. In conclusion, immunohistochemistry with monoclonal anti-SAA antibody Reu.86.2 is an easy and highly reliable method to detect amyloid AA in rectal biopsies.

Differential Influence of Glucocorticoids on CRP and SAA Serum Levels in Rheumatoid Arthritis and Polymyalgia Rheumatica

Aim of this study in patients with rheumatoid arthritis (RA) and polymyalgia rheumatica (PMR) was to investigate the differential effects of immunosuppressive therapy on the serum levels of C-reactive protein (CRP) and serum amyloid A (SAA), two acute-phase proteins. CRP and SAA were both quantified by ELISA. In a cross sectional study of RA patients, prednisolone therapy (median 7.5 mg daily) was associated with a higher SAA/CRP ratio compared to untreated patients (median value 1.67 vs. 0.63, p>0.005). Azathioprine did not show a similar effect on this SAA/CRP ratio. A longitudinal study in RA patients showed also a rise of the SAA/CRP ratio after the initiation of low dose (median 7.5 mg) prednisolone therapy (p>0.05). In a cross sectional study of PMR patients a positive correlation was observed between the daily dose of prednisolone and the SAA/CRP ratio (r=0.65, p>0.001). A longitudinal study in PMR patients showed a rise of the SAA/CRP ratio after the start of high dose (median 40 mg) prednisolone (p>0.05). Conclusion: Although glucocorticoid treatment suppresses both CRP and SAA synthesis, this effect is more pronounced for CRP than for SAA. This may have consequences for the choice of drug treatment and for the evaluation of its effect in diseases complicated by AA amyloidosis.

Clinical Evaluation of Osteoarthropathy in 43 Patients on Haemodialysis, Using Cuprophane Membranes for ≥ 5 Years

43 stable patients on chronic haemodialysis (using cuprophane membranes exclusively ≥ 5 years) were evaluated for symptoms of dialysis associated osteoarthropathy. Median time on dialysis was 120 months (range 60–260). The 16 women and 27 men were included with a median age of 56 years (range 33-76). Shoulderpain was seen in 29 patients (67%) with an actuarial risk of 50% at 134 months. Objective testing showed in 30 patients (70%) some loss of function; of whom 19 (46%) were positive on questioning. Carpal tunnel syndrome (CTS) was present in 16 patients (37%) with a 50% risk at 184 months. Arthritis was seen in 19 patients (44%). Radiological abnormalities were observed in 24 patients (56%). The number of serious symptoms increased with the time on dialysis. Synovial biopsies in 8 out of 11 patients with three serious symptoms established β2M-amyloidosis. Amyloidosis of the β2M-type was found in synovia, small vessels and to a lesser degree in the heart in a post-mortem examination. In conclusion symptoms of osteoarthropathy were frequently present in this group. The number of serious symptoms was related with the time on dialysis. β2M-amyloidosis was seen in 8 patients. This β2M-amyloid is one of the types of systemic amyloidosis.

Serum Amyloid a and C-Reactive Protein in the First 4 Weeks after Liver Transplantation

SAA and CRP serum levels were monitored for 4 weeks after liver transplantation (Tx) in 10 patients. Median age of these 6 women and 4 men was 41 years (range 17-55). Therapy consisted of high dose glucocorticoids with azathioprine. One week after Tx a biopsy was taken to detect acute rejection. SAA and CRP were measured by ELISA. Median SAA level before Tx was 2.5 mg/1 and median CRP 9.4 mg/1. Top SAA and CRP levels were seen after 1 to 3 days. The severity of acute rejection did not correlate with SAA and CRP levels 2,3,7 and 14 days after Tx. Basal reference values for “healthy” (N=19) patients 1-3 years after Tx were calculated: Median SAA 2.1 mg/1, 95% upper limit 6.2 mg/1. Median CRP 0.35 mg/1, 95% upper limit 3.7 mg/1. SAA values were higher than basal values of 50 healthy controls (p>0.0001), while CRP values did not differ. In conclusion, the diseased liver was deficient in the production of CRP and especially SAA. CRP and SAA levels did not correlate with the acute rejection found one week after Tx. Basal reference values of “healthy” patients years after Tx showed a modest elevation of SAA levels compared to healthy controls. This elevation of SAA in combination with normal CRP might be caused by maintenance therapy with glucocorticoids, although subclinical rejection cannot be excluded.

Crp and SAA Serum Levels in Chronic Active Hepatitis

C-reactive protein (CRP) and serum amyloid A (SAA) were studied in chronic active hepatitis (CAH), a disease of the principal site of CRP and SAA production. CRP and SAA levels were compared with histological severity and levels of Cholinesterase (CHE), a representative of liver synthesis capacity.