Anti-serum amyloid component P antibodies in patients with systemic lupus erythematosus correlate with disease activity.

OBJECTIVE: To determine the presence of raised titres of anti-serum amyloid P component (SAP) antibodies in patients with systemic lupus erythematosus (SLE) and to evaluate their correlation with clinical disease by the SLEDAI and clinical manifestations. METHODS: 452 samples were screened for raised anti-SAP antibody titres by an ELISA. Clinical measures and SLEDAI scores were independently reviewed from medical records. 21 serial samples from 7 patients with SLE were assessed for a change in anti-SAP antibody titres after treatment. RESULTS: Raised anti-SAP antibody titres were detected in 145/328 (44%) SLE samples. In 112 randomly selected samples, 69/112 (62%) patients had raised anti-SAP antibodies and anti-dsDNA antibody titres, whereas only 32/112 (28%) had raised anti-dsDNA antibody titres without raised anti-SAP antibody titres. The mean titre of anti-SAP antibodies in patients with active disease was higher than in patients with inactive disease and controls. SLEDAI scores, assessed in 54 patients, were raised in 26/31 (84%) patients with raised anti-SAP antibody titres. A SLEDAI score >or=8 was found in 16/31 (52%) patients with raised anti-SAP antibody titres but in only 5/23 (22%) patients without raised titres. No specific pattern of disease was detected in patients with or without raised titres of anti-SAP antibodies. Serial sampling from patients with active SLE and raised anti-SAP antibody titres showed that anti-SAP antibody titres decreased after treatment and correlated with clinical improvement. CONCLUSION: Raised anti-SAP antibody titres detected in patients with SLE correlate with disease activity and decrease with improvement of clinical disease, and thus may serve as an additional prognostic marker.

Severe disease in patients with rheumatoid arthritis carrying a mutation in the Mediterranean fever gene.

BACKGROUND: Pyrin is a newly recognised intracellular regulator of inflammation, and mutations in MEFV, the gene encoding pyrin, are the cause of familial Mediterranean fever. OBJECTIVE: To determine if known mutations of MEFV are associated with rheumatoid arthritis (RA) morbidity or can modify RA severity. METHODS: The frequency of the three most common MEFV mutations: M694V, V726A, and E148Q, was determined in 98 Israeli patients with RA (74 women, 24 men) and compared with that in 100 healthy subjects matched for origin. RA severity was determined using a new clinical score of 126 grades. The median severity score of mutation carrier and non-carrier groups was compared after confounding measures were eliminated by logistic regression. RESULTS: 17/98 (17%) patients with RA (all women) were heterozygous for common MEFV mutations, predominantly E148Q (12 patients), and one patient was homozygous for the V726A mutation. The overall mutation rate was comparable between patients with RA and healthy subjects. Patients carrying a mutation had a higher median severity score than the non-carrier group (42 v 29, p = 0.0005). The logistic regression model assigned a 15-fold odds ratio for severe RA in carriers, after adjusting for sex, presence of rheumatoid factor, age at onset, and disease duration (n = 97, p = 0.01, 95% CI 1.74 to 128). CONCLUSION: MEFV, and particularly the E148Q mutation, is an independent modifier of the clinical manifestations of RA. This is the second Th1-type autoimmune disease in which MEFV mutations have been shown to aggravate the clinical status.

An ancillary tool for the diagnosis of amyloid A amyloidosis in a variety of domestic and wild animals.

Immunohistochemistry, the standard method for diagnosing amyloid A (AA) amyloidosis, is limited in animals because it requires a large array of animal-specific anti-AA antibodies, not commercially available. The Shtrasburg method (SH method) is a highly specific and sensitive technique, helping in the diagnosis and determination of AA amyloidosis in humans. The aim of this study is to determine whether the SH method is applicable in the diagnosis of AA amyloidosis in a variety of animals. Tissue samples were obtained from animals suffering from spontaneous or experimentally induced AA amyloidosis (mice, hamsters, guinea pigs, cheetahs, cats, cows, ducks, a dog, a goose, a chicken, and a turaco). Detection of the amyloid and quantitative evaluation were performed using Congo red staining, and specific AA typing was performed by the potassium permanganate technique. The studied tissues were subjected to the SH method, which confirmed the AA nature of the amyloid deposit, by displaying in polyacrylamide gel electrophoresis protein bands consistent with the molecular weight of the species-specific AA, in all the animals examined, except mice, hamsters, and guinea pigs. N-terminal analysis of these bands corroborated their AA origin. We conclude that the SH method may be used as an ancillary simple tool for the diagnosis of AA amyloidosis in a large number of domestic and wild animals. Moreover, our findings further increase the feasibility of applying this method in humans.

A possible favorable effect of colchicine in IgA nephropathy in a carrier of a MEFV mutation.

IgA nephropathy is the most common primary glomerulopathy. Currently, no satisfactory treatment is available and as a result, a significant proportion of affected patients progress to end-stage renal disease. We present a patient with IgA nephropathy in whom continuous colchicine treatment induced remission, which has lasted for 22 years. The patient was a carrier of a mutation in the FMF gene (MEFV). This case raises hopes for a better prognosis in at least one subgroup of IgA nephropathy, consisting of patients who happen to be heterozygous carriers of MEFV mutations.

Increased propensity for amyloidogenesis in male mice.

BACKGROUND: The male sex is a risk factor for reactive amyloidogenesis in several disease entities. Environmental, socioeconomic or genetic factors may underlie this male preponderance. This study was aimed at discovering whether male sex predisposes to reactive amyloidosis also in mice and to elucidate some of the hormonal associations of this risk. METHODS: Male and female Swiss mice were subjected to an established amyloid induction protocol and the amount of their splenic amyloid was determined and compared. The effect of estrogen, progesterone, testosterone and adrenalin on amyloidogenesis was studied in both sexes by administering these hormnones during amyloid induction and comparing the amount of splenic amyloid of the study mice with the control mice which received the amyloid induction protocol alone. RESULTS: Amyloid deposition appeared to be more abundant in male mice. This gender difference was not associated with any of the 3 sex hormones tested. Despite an expected increment, adrenalin caused an attenuation of amyloid deposition. CONCLUSIONS: The preferential expression of reactive amyloidosis in male mice seems to be unrelated to the common sex hormones. Increased production of other hormones such as adrenalin, or perhaps an augmented susceptibility to their effect, may cause gender differences by suppressing female amyloidogenesis. Our study favors the hypothesis of genetic predisposition as the mechanism leading to sex differences in amyloidogenesis. Further validation of our findings in gonadal ablated models and other amyloid induction protocols is warranted.

Micropurification techniques in the analysis of amyloid proteins.

This review describes the different microtechniques developed for the extraction and purification of amyloid proteins from small specimens of fresh and formalin fixed tissues. These procedures differ with respect to solvent type, extraction conditions, and protein purification strategy. The advantages and disadvantages of the different microtechniques are discussed by taking into consideration tissue type (fresh of fixed) and size, amyloid type, and its content in the tissue. The review demonstrates the applicability of these techniques for the immunochemical and chemical characterisation of amyloid in different clinical forms of amyloidosis and in experimental small animal models. The clinical value of the applied microtechniques and their importance in the study of the pathogenesis of amyloid related diseases are outlined.

Laparoscopic splenectomy for torsion of wandering spleen associated with celiac axis occlusion.

BACKGROUND: Wandering spleen is a spleen lacking its normal ligamentous attachments, and thus subjected to free movement in the abdominal cavity, and even torsion around its pedicle. Surgical treatment includes either fixation (splenopexy) or resection (splenectomy). Both procedures can now be accomplished using the laparoscopic approach. METHODS AND RESULTS: We describe a case of a torsion of a wandering spleen, leading to recurrent episodes of abdominal pain, and eventually to splenic ischemia, necessitating splenectomy. The diagnosis was complicated by associated angiographic findings of celiac axis occlusion, possibly by median arcuate ligament compression. Laparoscopic splenectomy was successful, and led to complete resolution of symptoms. CONCLUSIONS: Although a rare condition, wandering spleen can be diagnosed accurately by imaging studies, mainly CT scan and angiography. Nowadays, the laparoscopic approach is preferred and enables the surgeon to perform either splenopexy or splenectomy, depending on the vascular status of the spleen.

Light chain amyloidosis of the urinary bladder. A site restricted deposition of an externally produced immunoglobulin.

AIMS: To identify the amyloid protein in a patient with amyloidosis localised to the urinary bladder, and to see whether subtyping of the protein by sequence analysis increases the understanding of the selection of the urinary bladder as the site of amyloid deposition. METHODS: A patient with gross haematuria and a congophilic mass in his urinary bladder was evaluated further. Characterisation of the amyloid protein was performed using conventional histological and immunohistochemical methods. Determination of the N-terminal amino acid sequence of the amyloid protein was performed using protein sequencers. RESULTS: The patient's history, physical examination, and laboratory evaluation excluded the involvement of other organs, justifying a diagnosis of amyloidosis localised to the urinary bladder. Histological and immunological studies showed that the amyloid protein deposited in the urinary bladder of the patient was probably of the amyloid light chain type. No plasma cells or lymphocytes were seen in sections of the urinary bladder and lower ureter adjacent to the amyloid deposits. Molecular analysis showed the sequence NFMLTQPHSISGSPG, which assigned the amyloid protein to either the Vlambda(I) or the Vlambda(VI) immunoglobulin (Ig) light chain families. CONCLUSIONS: The findings suggest that the amyloid protein in this patient originated outside the urinary bladder. The heterogeneity of the Ig proteins in known cases of amyloidosis of the lower urinary tract suggests that the amino acid residues, which determine the Vlambda subtyping, have no major role in restricting the deposited protein to the urinary bladder.

Common MEFV mutations among Jewish ethnic groups in Israel: high frequency of carrier and phenotype III states and absence of a perceptible biological advantage for the carrier state.

Familial Mediterranean fever (FMF) is an autosomal recessive disease, characterized by recurrent attacks of fever and inflammation of serosal membranes and gradual development of nephropathic amyloidosis. The recent cloning of the FMF gene (MEFV) and identification of disease-associated mutations in most patients made the direct determination of FMF carrier frequency feasible. The aim of the present study was to investigate the carrier rate of the most common MEFV mutations among different Jewish ethnic groups in Israel. Further, an attempt was made to elucidate the possible biological advantage that the heterozygote state may confer. Three hundred Ashkenazi, 101 Iraqi, and 120 Moroccan Jews were screened for the E148Q, V726A, and M694V mutations (at least two most common mutations per group), with a resulting overall carrier frequency in the respective ethnic group of 14%, 29%, and 21%. No difference in morbidity between Ashkenazi carriers and non-carriers of MEFV mutations was discerned, although an excess of febrile episodes in carriers of the V726A and in carriers of either V726A or E148Q was evident (P < 0.02 and P < 0.05, respectively). The frequency of subjects with two MEFV mutations but not expressing FMF (phenotype III) was 1:300 in Ashkenazi Jews and 1:25 in Iraqi Jews, exceeding the reported rate of overt FMF in these ethnic groups by 40-240 fold. These results affirm the high carrier rate among the studied Jewish ethnic groups in Israel and suggest that most subjects with FMF mutations are unaffected.

Inhibition of the second phase of amyloidogenesis in a mouse model by a single-dose colchicine regimen.

Amyloidogenesis consists of two stages. In the first, amyloid enhancing factor (AEF) is generated, and in the second, deposition of amyloid fibrils occurs. Colchicine is a known inhibitor of amyloidosis of familial Mediterranean fever (FMF) and of mouse experimental amyloidosis, but the timing and mechanism of its effect are still unclear. The aim of this study is to determine whether colchicine inhibits the second phase of amyloidogenesis and to study the time correlate of such an effect. To that end, amyloid was induced in Swiss male mice with AEF and AgNO(3) (an inflammatory stimulus), a method that skips the first phase of amyloidogenesis. Two amyloid induction protocols were used: a standard protocol, in which AEF and AgNO(3) were administered concurrently, and a prolonged protocol, in which the administration of AgNO(3) was delayed by 24 hours or 7 days. To study the inhibitory effect of colchicine on the second phase of amyloidogenesis, a single dose of colchicine (30 microg) was injected intravenously before, during, or after administration of AgNO(3) in both the standard and prolonged amyloid induction protocols. The amount of amyloid deposition in the spleens was determined with the crush-and-smear technique and a 5-grade scale. Colchicine was found to inhibit the second phase of amyloidogenesis. Its best effect was achieved when administered 48 hours after initiation of AgNO(3) injections. The pattern of colchicine-inhibition-in-time in the standard and the prolonged amyloid induction protocols was similar, indicating that colchicine exerts inhibition through its effect on the inflammatory stimulus (AgNO(3)). These findings suggest that (1) colchicine suppresses amyloidogenesis in the late (second) stage and that (2) this suppression is possibly related to the anti-inflammatory effect of colchicine.

Prevalence of ischemic heart disease in patients with familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever is a genetic disorder manifested by recurrent attacks of peritonitis, pleuritis and arthritis, and characterized by clinical, histological and laboratory evidence for localized and systemic inflammation. Colchicine treatment usually prevents the attacks and the associated inflammation. Inflammation may play an important role in the initiation and progression of atherosclerosis and ischemic heart disease. OBJECTIVE: To study the effect of inflammation and its prevention on the occurrence of IHD, using FMF as a model. METHODS AND PATIENTS: We studied the presence of IHD and its risk factors in 290 FMF patients aged 40 years or more, and in two control groups--233 spouses of the FMF patients, and 126 patients with inflammatory diseases obtained from other outpatient clinics, FMF patients were also compared with age and gender-matched individuals from the population reference data of the Israel Ministry of Health. RESULTS: The prevalence of IHD in FMF patients was significantly lower than in the group of controls from other outpatient clinics (15.5% vs. 30.2%, P < 0.05) and comparable with their spouses (11.2%) and with the matched general population in Israel (16%). CONCLUSIONS: These findings suggest that despite the evidence of recurrent inflammation, colchicine-treated FMF patients are not more predisposed to IHD than the normal population.

A single mutated MEFV allele in Israeli patients suffering from familial Mediterranean fever and Behçet's disease (FMF-BD).

Although familial Mediterranean fever (FMF) is an autosomal recessive disorder, preliminary partial mutation analysis suggested that about 60% of FMF patients, who also suffer from Behçet's disease (FMF-BD), have only a single mutated FMF gene (MEFV). In this study, the possibility that patients with FMF-BD may indeed be carriers of a single mutated MEFV is further analysed. The presence of mutations in the coding region of MEFV of eight patients with FMF-BD, representing six families with 47 members, was determined by sequencing. A possible role for the non-carrier chromosome and for BD in the expression of FMF in patients with a single mutated MEFV allele was determined by analysing the association between these variables and the presence of FMF in heterozygous kin. Sequence analysis revealed that all eight patients had indeed only one mutation in the coding region of MEFV. The patients' non-carrier chromosomes converged into three different MEFV haplotypes and were shared by heterozygous unaffected kin in five of six families. BD was found in 10 of 11 carriers with FMF vs one of 16 carriers without FMF (P < 0.001). These results suggest that FMF may be expressed in individuals harbouring only one coding mutation in MEFV. The findings argue against a role for the non-carrier chromosome in the induction of FMF, and suggest that the FMF phenotype in this cohort was associated with the simultaneous presence of BD. These findings may mirror a more generalised rule, that FMF may be precipitated in carriers of a single mutated FMF gene by factors unrelated to the other MEFV allele.

Micro-method to isolate and purify amyloid proteins for chemical characterization.

The amyloidoses represent a heterogeneous group of disorders characterized by the pathologic deposition as fibrils of at least 20 different precursor molecules. To establish definitively the specific type of amyloid protein contained in fibrillar deposits, such material must be extracted, purified, and subjected to amino acid sequence analysis. Heretofore, the chemical identification of amyloid components has required gram quantities of tissue. Given the often-limited amounts of sample available, e.g., that derived from diagnostic needle biopsies, we have developed a micro-method to isolate and purify amyloid from minute tissue specimens. The procedure involves micro-extraction of the amyloid with subsequent purification by SDS-PAGE, electroblotting onto PVDF membranes, excision and elution of amyloid protein-related bands, and reversed phase HPLC. Chemical and immunologic studies of isolated amyloid components have demonstrated the purity achieved with this technique and have provided information on the molecular mass, heterogeneity, and immunoreactivity of the amyloid. Further, using this methodology, it has been possible to obtain sufficient material for amino acid sequencing and thus to establish unequivocally the chemical and molecular composition of the fibrillar deposits. Our microtechnique has clinical import and also is applicable to analyses of the amyloid found in experimental small animal models of these disorders.

Abdominal CT findings in nephropathic amyloidosis of familial Mediterranean fever.

To evaluate the abdominal CT features of reactive amyloidosis, abdominal CT scans of 20 patients with amyloidosis of familial Mediterranean fever (FMF) were reviewed and compared with abdominal CT scans of 2 control groups: 22 patients with chronic renal failure (CRF) due to non-amyloidotic kidney diseases and 40 patients with normal kidney function. The kidney size of patients with amyloidosis of FMF were found to vary during the course of the disease from normal or slightly larger than normal at the proteinuric phase, to smaller than normal and comparable to kidney size in CRF, at the uremic stage. Compared to kidney disease of other causes, more patients with FMF-amyloidosis had dense kidneys with coarse parenchymal calcification and calcification in other abdominal organs. Patients with FMF-amyloidosis had fewer aortic calcifications than patients with non-amyloidotic kidney disease. These findings suggest that kidney disease of reactive amyloidosis may have abdominal CT findings distinguishing it from other types of kidney diseases.

Pregnancy and amyloidosis: II. Suppression of amyloidogenesis during pregnancy.

The observation of a deleterious effect of pregnancy on kidney function in amyloidosis of familial Mediterranean fever suggests that pregnancy may enhance amyloidogenesis. To determine whether pregnancy may indeed affect amyloidogenesis, pregnant mice were made amyloidotic by administration of amyloid-enhancing factor (AEF) and AgNO3 at different points in time from conception, and amyloid- deposition was studied with the crush-and-smear technique. A possible effect of exogenous female sex hormones (beta-estradiol and progesterone) on amyloidogenesis was studied by administration of these hormones during amyloid induction in nonpregnant female mice. Amyloidogenesis was found to be significantly suppressed in mice during pregnancy. The reduction was possibly related to the effect of pregnancy on the inflammatory stimulus (AgNO3) and not on the administered AEF. Exogenous estrogen and progesterone failed to inhibit amyloidogenesis in nonpregnant mice. These findings suggest that pregnancy may suppress amyloidogenesis in mice. The suppression is caused by an anti-inflammatory effect of pregnancy. Estrogen and progesterone are probably unrelated to this finding.

Thromboembolism in a patient with transient eosinophilia and thrombocytopenia.

A 24-year-old woman with an unremarkable medical history who developed bilateral deep venous thrombosis and pulmonary emboli is presented. Associated findings were severe eosinophilia and moderate thrombocytopenia. Since the major acquired and hereditary thrombogenic disorders were ruled out in this case (including antiphospholipid syndrome and heparin-induced thrombocytopenia), we believe that the severe eosinophilia per se could be the pro-coagulant factor leading to thrombosis and embolism in our patient. The role of eosinophilia in thrombosis is discussed.

Protracted febrile myalgia of familial Mediterranean fever. Mutation analysis and clinical correlations.

Protracted febrile myalgia (PFM) is a rare form of vasculitic disease that affects patients with familial Mediterranean fever (FMF). Mutation analysis performed in 15 patients who suffered from this disorder showed that 9 of the 15 patients were homozygous for M694V. FMF in these 9 patients was associated with more severe symptoms compared to a group of 30 M694V homozygous FMF patients without PFM.