[ANCA(antineutrophil cytoplasmic antibodies)-associated vasculitis in a man with extreme fatigue, fever and progressive renal dysfunction]. / Vasculitis geassocieerd met antineutrofielencytoplasmatische antistoffen (ANCA) bij een man met ernstige vermoeidheid, koorts en een progressieve nierfunctiestoornis.

A 55-year-old man, with no previous history, presented with extreme fatigue and fever and was admitted to hospital. He had progressive renal dysfunction and his serum anti-neutrophil cytoplasmic antibodies (ANCA) were markedly elevated. Renal histology was consistent with ANCA-associated vasculitis. The patient was successfully treated with cyclophosphamide and prednisolone. The classification and management of the ANCA-associated vasculitides are described. The classification was guided by the clinical presentation, serology and results of tissue biopsies. The ANCA inflammation had affected the middle sized and small vessels of especially the upper and lower airways, and the kidneys. The antibodies were directed at proteinase-3 (PR3) or myeloperoxidase (MPO). PR3-ANCA is predominantly found in Wegener's granulomatosis, while MPO-ANCA is related to microscopic polyangiitis. Tissue studies showed granulomatous inflammation of the airways which is typical of Wegener's disease. This type of inflammation is absent in microscopic polyangiitis. The initial treatment schedule consists of prednisone 1 mg/kg daily and oral cyclophosphamide 2 mg/kg daily. In the remission phase, the cyclophosphamide is replaced by azathioprine. It is not yet known how long maintenance treatment should be continued and which parameters have prognostic value.

Iron mobilization from ferritin by superoxide derived from stimulated polymorphonuclear leukocytes. Possible mechanism in inflammation diseases.

During inflammation, the superoxide anion (O-2) and hydrogen peroxide (H2O2) are produced by stimulated polymorphonuclear leukocytes and macrophages. The toxic effects of these reactive oxygen intermediates increase when traces of iron are present, because iron catalyzes the formation of the hydroxyl radical (OH.). Partially saturated iron-binding proteins, such as transferrin and ferritin, are unable to catalyze OH. formation in vitro. Mobilization of iron from these proteins is necessary for iron stimulation of OH. formation. This paper reports that stimulated polymorphonuclear leukocytes mobilize iron from human and horse ferritin, but not from human transferrin. Iron release from ferritin depends on O-2 because it can be prevented by the addition of superoxide dismutase. Catalase and dimethylsulfoxide have no inhibitory effect on iron mobilization. The efficiency of the iron release increases at low levels of O-2 production. Only O-2 produced by granulocytes is sufficient for iron mobilization, because solid potassium superoxide is also able to release iron from ferritin. We propose that this reaction may potentiate the formation of the OH. radical in inflammatory states.

[Progressive multifocal leukoencephalopathy in a patient following long-term immunosuppressive therapy for systemic lupus erythematosus]. / Progressieve multifocale leuko-encefalopathie bij een patiënte na langdurige immunosuppressieve behandeling wegens lupus erythematodes disseminatus (SLE).

A 41-year-old woman who had suffered from systemic lupus erythematosus (SLE) for 22 years presented with signs of neurological deficits. CT-scanning of the brain revealed hypodense lesions that suggested cerebral infarction due to vasculitis in SLE. However, in spite of intensified immunosuppressive therapy, she showed rapid neurological deterioration. After extensive, additional examinations and tests, the diagnosis was finally changed to progressive multifocal leukoencephalopathy, caused by an opportunistic infection by the JC polyomavirus. Neurological and psychiatric symptoms frequently occur in patients with SLE. The differential diagnosis of these symptoms in SLE is extensive and includes, on the one hand, primary neurological and psychiatric diseases related to direct involvement of the nervous system by SLE, and on the other hand, secondary syndromes arising as a result of complications of the SLE or the immunosuppressive treatment. Opportunistic infections are often an important secondary cause of neurological and psychiatric syndromes in patients with SLE. The clinical symptoms and radiological cerebral signs are non-specific and usually do not suffice to differentiate between the various syndromes. Since each syndrome requires its own specific clinical approach and treatment, extensive diagnostics are mandatory before the diagnosis 'cerebral lupus' can be made and immunosuppressive therapy can be started or intensified.

Superoxide dependent iron release from ferritin in inflammatory diseases.

Convincing evidence is presented that oxygen free radicals are involved in the pathogenesis of rheumatoid arthritis (RA). Superoxide is produced by polymorphonuclear leucocytes (PMN) in synovial fluid and by macrophages in the synovial membrane. Tissue damage typical for free radical attack is detected in RA. No absolute deficiency of protective factors is found in RA compared to controls, but the available protection is insufficient to cope with all radicals formed. The toxicity of superoxide is increased by iron. It is doubtful whether a low molecular weight iron pool is present. Superoxide is able to release iron from ferritin, providing a suitable source of iron, for the formation of hydroxyl radicals. This new pathogenetic mechanism stimulates to the application of iron chelators in the treatment of RA. Preliminary results with desferrioxamine were disappointing because of serious side-effects. Hopefully in the future intra-articular injection of iron chelators with better pharmacodynamics will be possible. The interaction of free radicals and ferritin is probably also involved in the pathogenesis of other inflammatory diseases such as systemic lupus erythematosus, hepatitis, and haemochromatosus.

Production of monoclonal antibodies against inactivated alpha 1-antitrypsin. Cross-reactivity with complexed alpha 1-antitrypsin and application in an assay to determine inactivated and complexed alpha 1-antitrypsin in biological fluids.

15 different monoclonal antibodies (mcAbs) have been raised against the cleaved (inactive) form of the serpin alpha 1-antitrypsin (AT). In initial experiments these mcAbs were analysed for their ability to bind the native and the cleaved form of this inhibitor: eight of the 15 mcAbs appeared to react predominantly with cleaved AT. Additional experiments with mixtures of purified native AT, AT complexed to neutrophilic elastase and inactivated AT revealed that all mAbs that preferentially reacted with inactivated AT also bound to complexed AT. Using two of the mcAbs against inactivated AT a quantitative and sensitive sandwich-type radioimmunoassay was developed to determine levels of proteolytically inactivated AT in biological fluids. With this assay increased levels of inactivated AT were found in synovial fluid from patients with rheumatoid arthritis corresponding to about 2.4% (range 0.3-11%) of total AT. Approximately 10% of this inactivated AT appeared to consist of AT complexed to neutrophil elastase. The mcAbs described here further illustrate the structural resemblance between the complexed and cleaved forms of AT. In addition, these mcAbs appear to be useful tools for the study of AT in human disease.

A new ELISA for the detection of anti-heparan sulfate reactivity, using photobiotinylated antigen.

Autoantibodies reacting with a great variety of autoantigens are characteristic for the autoimmune disease systemic lupus erythematosus (SLE). Although reactivity with heparan sulfate (HS) in sera of patients with SLE is found in association with the occurrence of nephritis, the aetiological significance of this association is not clear. The assay which is generally used to measure anti-HS reactivity is subject to false-positive results, as a consequence of the binding of negatively charged moieties within immune complexes to the precoat employed (protamine sulfate). Therefore, we have developed a new ELISA in which photobiotinylated HS is efficiently and reproducibly bound to streptavidin-coated wells. We compared the new ELISA with the classical anti-HS ELISA by testing culture supernatants of 20 murine monoclonal antibodies (mAb) to DNA (containing free anti-DNA and anti-DNA/nucleosome immune complexes) and preparations of these mAb (containing only free anti-DNA), purified under dissociating conditions. In the classical anti-HS ELISA, 14 out of 20 of the culture supernatants reacted positively with HS; after purification no reactivity remained. The discrepancy must be due to anti-DNA/nucleosome immune complexes present in the culture supernatants. In the new ELISA only four out of 20 culture supernatants and one of the purified preparations reacted with HS. This latter reactivity is probably not specific, since this mAb also reacted with streptavidin alone. To find out whether there is a correlation between the occurrence of nephritis and anti-HS reactivity, measured in this new anti-HS ELISA, we tested sera of patients with a renal- or non-renal exacerbation of SLE in the newly developed anti-HS ELISA. We observed a correlation between anti-HS reactivity and nephritis.

Application of a monoclonal antibody against a neoepitope on activated C4 in an ELISA for the quantification of complement activation via the classical pathway.

In order to study the activation of the complement system via the classical pathway we have attempted to raise antibodies specific for C4 activation products. Of 20 mouse monoclonal antibodies (mAbs) obtained, one appeared to react with an activation dependent epitope exposed on the activation products C4b, C4bi, C4c (C4b/c) as well as on iC4, but not on native C4. Using this antibody as a capture antibody and polyclonal biotinylated antibodies against C4 as detecting antibodies we developed an ELISA for the quantification of C4b/c in biological fluids. The lower limit of detection was approximately 0.025 nmol C4b/c per litre. Mean C4b/c levels in plasma samples collected from healthy volunteers in tubes containing 10 mM EDTA and 0.05% (w/v) polybrene, final concentrations, appeared to be 30 nmol/l. The potential of the ELISA procedure for evaluating complement activation in clinical samples was demonstrated.

Clinical evaluation of a modified ELISA, using photobiotinylated DNA, for the detection of anti-DNA antibodies.

The measurement of anti-dsDNA antibodies is important for the diagnosis and the follow-up of patients with systemic lupus erythematosus (SLE). For routine detection of anti-dsDNA, the Farr assay and the immunofluorescence technique (IFT) on Crithidia luciliae proved to be very useful. The anti-dsDNA ELISA is not used for routine purposes in our institute since it is flawed by false-positive results due to binding of negatively charged (immune) complexes to the employed precoat (protamine sulphate). Recently, a new anti-dsDNA ELISA has been described in which photobiotinylated dsDNA is coated to streptavidin coated plates. To investigate whether this modified ELISA is more specific than the classical anti-dsDNA ELISA, we tested sera of patients with SLE (n = 51), myasthenia gravis (MG, n = 25), rheumatoid arthritis (RA, n = 25) and Sjögren's syndrome (SS, n = 23) and sera of healthy blood bank donors (BBD, n = 25). In both assays the sera of the SLE patients gave significantly higher values than the sera of healthy blood bank donors. In the classical ELISA, 84% of the sera from patients with RA and 28% of sera of patients with MG were found positive. For the modified assay the figures were 8% and 24%, respectively. This modified ELISA was further studied and clinically evaluated by comparing it with the classical anti-DNA ELISA and two other anti-DNA assays (Farr assay and IFT), using 500 sera sent to our institute for routine anti-DNA determination and sera of an additional 75 healthy blood bank donors. Quantitatively, both ELISAs showed the same high degree of correlation with the IFT. The modified ELISA gave a better correlation with the Farr assay than the classical anti-DNA ELISA. From our data we conclude that the ELISA using photobiotinylated DNA is a more reliable assay than the classical anti-DNA ELISA.

Characteristics of recovery from the euthyroid sick syndrome induced by tumor necrosis factor alpha in cancer patients.

Cytokines have been implicated in the pathogenesis of the euthyroid sick syndrome. Isolated limb perfusion (ILP) with recombinant human tumor necrosis factor alpha (rTNF) and melphalan in patients with melanoma or sarcoma is accompanied by high systemic TNF levels. We examined the prolonged effects (7 days) of ILP on thyroid hormone metabolism with respect to induction and recovery of the euthyroid sick syndrome in six cancer patients. After ILP, when the limb is reconnected to the systemic circulation, leakage of residual rTNF resulted in systemic peak levels at 10 minutes postperfusion followed by a parallel increase in plasma interleukin-6 (IL-6) and cortisol, with maximum levels at 4 hours (P < .05). A rapid decrease was observed at 5 minutes for plasma triiodothyronine (T3), reverse T3 (rT3), thyroxine (T4), and thyroxine-binding globulin (TBG) (P < .05), whereas free T4 (FT4) and T3-uptake showed a sharp increase, with peak levels at 5 minutes (P < .05). T3, T4, and TBG levels remained low until 24 hours after ILP In contrast, rT3 increased above pretreatment values to maximum levels at 24 hours (P < .05). Plasma thyrotropin (TSH) showed an initial decrease at 4 hours postperfusion (P < .05) but exceeded pretreatment values from day 1 to day 7 (by +94%+/-43% to +155%+/-66%, P < .05), preceding the recovery of T4 and T3 levels. T3 and rT3 returned to initial values at day 4. T4 and TBG levels recovered at day 2. T4 exceeded basal values at days 5 to 7 (P < .05). It is concluded that ILP with rTNF induces a euthyroid sick syndrome either directly or indirectly through other mediators such as IL-6 or cortisol. The recovery from this euthyroid sick syndrome is, at least in part, TSH-dependent, since the prolonged elevation of TSH values preceded and persisted during the normalization of T3 and the elevation of T4 levels. This biphasic pattern of induction of and recovery from the euthyroid sick syndrome may be a general feature of nonthyroidal disease. The euthyroid sick syndrome should be interpreted not only in relation to the presence of nonthyroidal diseases but also in relation to the recovery from these diseases.

Impaired erythropoietin responsiveness to the anaemia in rheumatoid arthritis. A possible inverse relationship with iron stores and effects of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one.

We investigated the serum erythropoietin (Epo) response in 11 rheumatoid arthritis (RA) patients without anaemia, 7 with RA and iron deficiency (ID) and 12 with RA and anaemia of chronic disease (ACD). In all patients the serum Epo was higher than in healthy subjects. Apparently this increase was insufficient to prevent anaemia in ID and ACD. Serum Epo correlated negatively with serum ferritin. Ten RA patients with ACD were treated with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). No obvious toxicity signs occurred after one week of treatment. It effectively released iron from iron stores. The Hb rise (in 70% of the patients) was correlated positively with an Epo increase and negatively with a serum ferritin decrease. We conclude that a serum Epo increase does not overcome ACD. Epo response might correlate inversely with iron stores. L1 treatment effectively chelates iron from iron stores. The effects of L1 on erythropoiesis and serum Epo and its safety need further substantiation after prolonged treatment in more RA patients.

Tear fluid analysis in primary Sjögren's syndrome.

Tear fluid analysis was performed in 44 patients with primary SS, 21 patients suspected of having primary SS in whom the syndrome had been excluded, and 24 control subjects. In the primary SS patients the tear fluid levels of lactoferrin and a1-antitrypsin were increased and the tear fluid levels peroxidase, lysozyme and amylase were decreased. However, a considerable overlap in the concentrations of all of the tested substances in the different groups was found and the measurement of these substances cannot be advocated for diagnostic use.

Progressive systemic sclerosis and IgA deficiency in a young man.

A 21-year-old man presented with an IgA deficiency and symptoms reminiscent of a systemic lupus erythematosus. After 7 years systemic sclerosis developed involving the lungs, oesophagus, central nervous system and reproductive system. Connective tissue disease and its relationship to IgA deficiency will be discussed.

Lymphocytotoxic antibodies in SLE: a review of the literature.

In a great majority of patients with systemic lupus erythematosus (SLE) lymphocytotoxic antibodies (LCA) are detected. The reported prevalence depends on the methods of detection and definition of test-positivity. The pathogenetic role of LCA remains unclear. Different reports show that LCA can have an influence on mononuclear cell function, e.g., on production of interferon, or specifically on T-cell function. In several studies T-cell specificity of LCA cannot be shown. Sometimes an overlap between LCA and anti-B2M antibodies is found, suggesting that LCA have anti-B2M activity. Anti-B2M activity might have an impact on T-cell as well as on B cell function. Next to the anti-B2M activity, binding to nuclear material is claimed. Investigating the role of LCA in respect to lymphopenia, a direct relationship cannot be found; however, LCA might induce interferon production, resulting in lymphopenia. Several studies show or claim a relationship between the presence of LCA and neurological manifestations in SLE patients; the results, however, remain questionable due to the difference in detection methods as well as in definition of central nervous system (CNS) involvement. In other studies, an increased incidence of LCA is reported in relatives of SLE patients as well. In addition, LCA are reported in quite a lot of other diseases such as rheumatoid arthritis, ankylosing spondylitis, malignancies and viral illnesses. This latter association has led to the assumption that LCA might have a virus-related origin.

Urine- and serum beta 2-microglobulin in patients with rheumatoid arthritis: a study of 101 patients without signs of kidney disease.

Serum levels and 24-hour urinary excretion of beta 2-Microglobulin (beta 2-M) was investigated in a group of 101 patients with rheumatoid arthritis (RA), without any other signs of renal disease in past or present, and in a comparable control group. Elevated 24-hour urinary beta 2-M excretion, due to renal proximal tubules dysfunction, was observed in 19% of the patients and not in the control group. There was a significant correlation with clinical signs of extra-articular RA. It is postulated that the observed increase may be an early symptom of renal involvement in RA. Elevated serum beta 2 levels, corrected for glomerular filtration rate, were observed in 44% of the RA patients and only in 3% of the control group and correlated with clinical signs of a more severe RA, as well as with increased 24-hour urinary beta 2-M excretion.

Pure red-cell aplasia in systemic lupus erythematosus.

A woman with SLE of six years duration developed severe anaemia which was characterized by severe bone marrow erythroid hypoplasia; no deficiencies could be found to explain this finding. Antibodies against erythroid progenitors are a likely cause of this anaemia, which completely resolved after transfusion of packed red-cells.

The role of erythropoietin in the anaemia of chronic disease in rheumatoid arthritis.

We reviewed studies on the role of erythropoietin (Epo) in the anaemia of chronic disease (ACD) in rheumatoid arthritis (RA). A relatively impaired Epo response to the anaemia was found in a number of studies although in others serum Epo level was the same as in other types of anaemia. Some arguments are found in favor of a reduced bone marrow-Epo sensitivity although these reflect results mainly from in vitro experiments. It is not yet established whether bone marrow macrophage Epo production is impaired in ACD. In two cases Epo administration to RA patients resulted in increased erythropoiesis. It was concluded that impaired Epo production or reduced bone marrow Epo sensitivity might be associated with ACD but it is not certain whether these factors are causally linked with ACD or side phenomena of RA disease activity. Future Epo treatment in RA and ACD will possibly solve this question.

Diagnostic significance of salivary levels of beta 2-microglobulin in Sjögren's syndrome.

To evaluate the diagnostic significance of salivary beta 2m in Sjögren's syndrome we measured salivary beta 2m levels in 19 patients with primary sicca syndrome (PSS), 15 with secondary Sjögren's syndrome (SSS) and compared the results with 20 normal healthy persons. We showed that beta 2m is specifically excreted in the saliva, because in normal saliva the concentration of beta 2m was unrelated to IgA levels. Also in normals, there was no relationship between serum and saliva concentrations of beta 2m. The mean saliva levels of beta 2m were increased in PSS (1.13 +/- 0.58) and SSS (1.39 +/- 0.69) compared with the levels in normals (0.53 +/- 0.22). The determination of beta 2m in the saliva can therefore be used as a noninvasive measurement for the confirmation of the diagnosis Sjögren's syndrome.

Polyarteritis nodosa in a young man, a ten-year delay in diagnosis.

We present a case history of a patient with polyarteritis nodosa (PAN). The first disease symptoms started when the patient was 6 years old and concerned mainly musculoskeletal complaints. The disease persisted into adulthood when, 10 years later, the diagnosis of PAN could be confirmed by histopathological examination.

Sarcoidosis presenting with severe thrombocytopenia and arthritis.

In this case report a patient with biopsy-proven sarcoidosis is described, presenting with joint symptoms and severe thrombocytopenia, due to immune-mediated platelet destruction.

Antinuclear antibody profiles in relation to specific disease manifestations of systemic lupus erythematosus.

UNLABELLED: The aim of the study was to investigate the prevalence of antinuclear and associated antibodies (anti-dsDNA, anticardiolipin, anti-RNP, anti-Sm, anti-SSA and anti-SSB) and/or combinations thereof in systemic lupus erythematosus (SLE) patients with respect to their diagnostic and pathogenetic significance. The prevalence of anti-dsDNA antibodies was strongly influenced by the selection criteria of the patient; the lowest prevalence was found in SLE patients with central nervous system (CNS) involvement; the highest prevalence in patients with nephritis. The results were also influenced by the different assays. Combinding different assays (Farr/PEG ratio) quantitative as well as qualitative differences could be shown between patients with nephritis (Farr/PEG ratio greater than or equal to 5) and with CNS involvement (Farr/PEG ratio less than 5). No difference in anticardiolipin antibody prevalence between the different SLE patient groups could be demonstrated. Regarding antibodies against RNP, Sm, SSA and SSB, the prevalence was found to be strongly influenced by the criteria used for patient selection. Only in CNS patients and association with anti-RNP and anti-SSB antibodies alone or in combination was found. In pleuropericarditis a weak association with RNP antibodies existed. IN CONCLUSION: studying the prevalence and possible pathogenetic significance of antibodies one should always consider patient selection criteria and the effect of the different assays used when analysing the results.