K. Andrassy

The value of indirect immunofluorescence and solid phase techniques for ANCA detection: A report on the first phase of an international cooperative study on the standardization of ANCA assays

This study describes the results of phase I of an international effort to develop and standardize assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). 12 sera, four of which were selected for their potential to cause problems in the detection of various ANCA specificities, were analyzed in the standard indirect immunofluorescence (IIF) test and in ELISAs for ANCA routinely performed in the seven participating laboratories. The IIF methodology differed with respect to the dilution of the serum being screened and the concentration of the conjugate used. Results from sera with high ANCA titers were similar, although the quantitative values could not be compared. In sera containing rheumatoid factor and anti-nuclear antibodies (ANA), ANCA-unrelated staining patterns were observed. Six antigen preparations were used in ELISA for the detection of cANCA. In ELISA with purified proteinase-3 all three cANCA sera were positive, but not anti-myeloperoxidase (MPO) or anti-lactoferrin (LF) positive sera. The other assays were less sensitive or gave inconsistent results. Various preparations of purified MPO and LF used in ELISA were readily recognized by anti-MPO and anti-LF positive sera. From this study it can be concluded that the IIF test, although performed with different methods, shows comparable results using strongly positive sera. In general solid phase assays for cANCA detection are not well standardized and need improvement although the purified proteinase-3 ELISA is possibly an exception. MPO and LF can be used in ELISA procedures for the detection of pANCA-related antibodies.

Comments on ‘KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease’

To the Editor: ‘KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease’1 fills a gap, now including very recent and new insights into the diagnosis and therapy of chronic renal disease. The structured presentation emphasizes this in various chapters coherently, including pediatric considerations. It is to be expected that these guidelines will represent the future basis for quick information on chronic kidney disease and not only for nephrologists. As happens in such an extensive overview, some well-known but older observations may have been lost owing to the quantity of new data, and in my opinion some statements are painfully missed for the diagnosis and treatment of chronic kidney disease.

Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T‐cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon‐γ (IFN‐γ) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II‐positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin‐2 (IL‐2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT‐specific T cells in a MHC class II‐restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T‐cell activation.

Hemostasis and thromboembolism in children with nephrotic syndrome: Differences from adults

Eleven of 204 children with nephrotic syndrome had thrombotic complications: arterial thrombosis in five, venous thrombosis in four, and pulmonary embolism in two. Fifty-one episodes of thromboembolism were recognized in 116 adult patients with nephrotic syndrome. Despite the lower incidence, thromboembolic complications tended to be more severe in children. In vitro indices of hemostasis and clinical evidence of thromboembolic complications were compared in children and adults. Antithrombin III concentrations and activities were abnormal in seven of 10 children, but in only two of 32 adults. In both groups, alpha 2-macroglobulin was elevated, but more markedly so in children. No evidence for circulating granulocyte-derived proteases (elastase/antielastase complexes) was noted in either group. Protein C was significantly elevated in children with nephrotic syndrome, but was normal in adults. Children also differed from adults with nephrotic syndrome in laboratory evidence of subthreshold disseminated intravascular coagulation (i.e., elevated soluble fibrinogen monomeric complexes and fibrin degradation products) and indicators of in vivo platelet activation (elevated beta-thromboglobulin). The more severe coagulation abnormalities in children may be linked to the more pronounced hypoalbuminemia.

European therapeutic trials in ANCA-associated systemic vasculitis: disease scoring, consensus regimens and proposed clinical trials EUROPEAN COMMUNITY STUDY GROUP ON CLINICAL TRIALS IN SYSTEMIC VASCULITIS ECSYSVASTRIAL (BMHl-Cr93-1078)

In previous phases of this project, proteinase 3 (PR3) and myeloperoxidase (MPO), the main antigenic target molecules of antineutrophil cytopiasmic antibodies, were isolated and applied in standardized ELISAs. In this study, standardized ELISAs with three PR3 preparations (from Copenhagen (CO), Raisdorf (RS) and Leiden (LF)) and one MPO preparation (from Copenhagen), were evaluated in a large retro-and prospective clitiical study. New patients (n=174) with primary systemic vasculitis (Wegeners granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive glomerulonephritis, classical PAN and Churg-Strauss Syndrome) were included. Retrospectively, another 190 patients were evaluated. Furthermore control sera were obtained from patients with other forms of vasculitis, glomerulonephritis or granulomatous diseases (disease controls, n = 184) and healthy donors (healthy controls, n = 728). All patients were categorized by a system based on clinical and histoiogical data. Patients were followed up for at least 1 year after diagnosis in order to evaluate a possible correlation between ANCA levels and disease activity. The sensitivity of the anti-PR3 assays for histologically proven WG was between 59% and 69% in new patients, with a sensitivity of 22% for the anti-MPO assay. Similar figures were found for patients with clinically suspected WG. This was comparable with the results of the IIF test. In MPA and IRPGN a larger percentage of patients had antiMPO antibodies than in WG. Only a few patients with PAN and CSS were investigated, and most of these were negative in the ELISAs. The specificity ofthe assays for disease controls was 89-91% for the anti-PR3 assays and 95% for the anti-MPO assay. In the healthy controls the specificity was 98-99%. The specificity of the IIF test was 97% for a cANCA pattern and 81 % for a pANCA pattern in disease controls. The combination of cANCA with anti-PR3 and pANCA with anti-MPO both had a specificity of 99%. Further details will be presented during the meeting, in addition to the results of a follow-up study with correlation ofdisease activity and ANCA level. From this study we can conclude that ELISAs using purified PR3 or MPO are not more sensitive than the IIF test. However, the anti-MPO assay is more specific for systemic vascuitis as compared to disease controls with related diseases. Furthermore, the combination of the IIF test with antigen-specific ELISAs is very specific for the diagnosis Wegetiers granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive gtomerulonephritis.

CRP levels in autoimmune disease can be specified by measurement of procalcitonin.

SummaryAutoimmune diseases (AID) are prone to infection particularly under immunosuppression. The differentiation of infection from active AID is often difficult. In order to specify the diagnostic value of measurement of procalcitonin (PCT) in AID 81 patients with anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis were analyzed, 27 with rheumatoid arthritis and 25 patients with systemic lupus erythematosus at various stages of the disease. Although PCT levels (95th percentile) were below 0.5 ng/ml in patients with active systemic lupus erythematosus and rheumatoid arthritis, the cutoff for normal values (95th percentile) in patients with active ANCA-positive vasculitis was 0.89. Therefore PCT levels of <1 ng/ml are recommended as cutoff for invasive infections in patients with ANCA-positive vasculitis. In view of the increased mortality under immunosuppression in patients with AID and additional bacterial infection the measurement of PCT is helpful when an infectious origin is suspected.

Transdifferentiation of polymorphonuclear neutrophils : acquisition of CD83 and other functional characteristics of dendritic cells

Polymorphonuclear neutrophils (PMN) are in the first line of defense against bacterial infections. They are considered to be end-differentiated cells undergoing constitutive apoptosis within hours after release from the bone marrow. During pathological events, however, their life span is extended in conjunction with morphological and functional alterations indicative of a transdifferentiation of mature PMN. To further characterize differentiated PMN, the alterations seen in vivo were reproduced by cultivating PMN of healthy donors with either γ-interferon, granulocyte/macrophage colony stimulating factor, or a combination thereof. Thus cultivated cells escaped from apoptosis, and protein synthesis was induced, notably of the major histocompatibility complex (MHC) class II antigens, CD80 and CD86. Moreover, CD83, thought to be specific for dendritic cells was synthesized, while typical markers of PMN, including CD66b, CD11a/CD11b/CD11c, CD15, CD18 were preserved. A profound alteration of both cellular morphology and of function was seen: the cultivated PMN lost their chemotactic activity but had acquired the ability to present to T-cells a peptide antigen in a MHC class II restricted manner. The data lead to the conclusion that mature PMN can differentiate further to cells with characteristics of DCs, thereby connecting PMN to the specific T-cell response.

Hypercoagulability in the nephrotic syndrome

ZusammenfassungDas nephrotische Syndrom (NS) ist die intern-medizinische Grunderkrankung mit dem höchsten Risiko an venösen (Unterschenkelvenen-Thrombose, Beckenvenen-Thrombose, Cava- und Nierenvenen-Thrombose ggf. mit Lungenembolie) und arteriellen (Herzinfarkt, Cerebralarterien-Thrombose, periphere arterielle Thrombose) thromboembolischen Komplikationen. Aufgrund neuerer hämostaseologischer Untersuchungen können beim NS Defekte des plasmatischen Gerinnungssystems, der Fibrinolyse und der Plättchenfunktion festgestellt werden. Infolge der globalen Synthesesteigerung hepatischer Exportproteine ist die Plasmakonzentration der meisten Gerinnungsfaktoren (speziell Faktor I, II, VII, VIII und X) gesteigert; infolge erhöhten renalen Verlustes ist die Plasma-Konzentration des Inhibitors Antithrombin III vermindert. Die Änderung der Plasma-Konzentration ist das Ergebnis gesteigerter hepatischer Synthese und/oder renalen Verlustes im Rahmen der Proteinurie. Hinweise für eine intravasale Gerinnung fanden sich — im Gegensatz zu Angaben der Literatur — in eigenen Untersuchungen nur selten. Die Plasminogenkonzentration ist vermindert, während die Gesamt-Aktivität der Plasmin-Inhibitoren erhöht ist. Allerdings ist die Alpha-1-Antitrypsin-Konzentration wegen gesteigerten renalen Verlustes meist vermindert, was jedoch durch erhöhte Konzentration anderer Inhibitioren, speziell Alpha-2-Makroglobulin und Alpha-2-Antiplasmin kompensiert wird. Obwohl im Urin Material gefunden wird, welches in der passiven Hämagglutination wie Fibrinspaltprodukte reagiert, zeigten neuere Untersuchungen, daß dieses Material Fibrinogensplatprodukte darstellt (nicht selektive glomeruläre Proteinurie) und nicht Fibrinspaltprodukte infolge lokaler oder systemischer Fibrinolyse. Die Plättchenzahlen sind normal oder mäßig erhöht und die Plättchen-Überlebenszeit ist geringfügig verkürzt. Hingegen lassen sich deutliche Abweichungen der Spontanaggregation sowie der ADP- und Kollagen-induzierten Aggregation nachweisen. Desgleichen ist die Plättchen-Aggregation durch Arachidonsäure gesteigert und die Malondialdehyd-Bildung erhöht. Die Plättchen nephrotischer Patienten weisen nach Zugabe von Albumin ein normales Aggregationsverhalten auf, was auf eine erworbene Funktionsstörung hinweist. Das wichtige Gebiet der Plättchenfunktion bei NS ist derzeit noch wenig erforscht. Aus den klinischen und hämostaseologischen Befunden wird die Forderung abgeleitet, bei Patienten mit nephrotischem Syndrom in jedem Falle Plättchen-Aggregationshemmer zu verabfolgen; bei Vorliegen venöser Thrombosen ist eine Langzeit-Antikoagulation mit Marcumar anzuraten.SummaryThe risk of thromboembolic complications in patients with the nephrotic syndrome (NS) is higher than in any other condition encountered in internal medicine. Such thromboembolic complications comprise venous thromboses (calf, thigh, renal vein) with or without pulmonary embolism and arterial thromboses (coronary thromboses, cerebral artery thromboses, peripheral arterial thromboses). Several defects of the plasmatic coagulation system, fibrinolysis and platelet function had been recognized in the nephrotic syndrome. Increased hepatic synthesis causes a rise of coagulation factors, I, II, VII, VIII, X and increased renal loss causes lowering of the plasma concentration of antithrombin III concentration. There is little evidence for DIC. Plasminogen concentration is diminished, whereas total antiplasmin activity is increased. Low alpha-1-antitrypsin concentration secondary to renal loss is outweighed by increased concentrations of other inhibitors especially alpha-2-macroglobulin and alpha-2-antiplasmin. The common presence of material in the urine reacting as fibrin degradation products with passive hemagglutination techniques appears to be proteolytically degraded fibrinogen excreted as a result of non-selective glomerular proteinuria. Platelet counts are normal or slightly elevated and platelet survival time is slightly, decreased. Definite abnormalities of spontaneous aggregation and ADP- or collagen-induced aggregation are demonstrable. Furthermore, arachidonic acid induced platelet aggregation and malondialdehyde formation by platelets of NS patients are increased. Addition of albumin to platelets of NS patients normalises platelet aggregation. This finding points to some acquired defect of platelet function.There is a clearcut relation between the risk of thromboembolic complications and plasma albumin concentration: thromboses are particularly frequent at plasma albumin concentrations below 2 g/dl. Consequently, it is suggested, that NS patients with plasma albumin <2 g/dl should be given platelet aggregation inhibitors prophylactically. If there is a history of venous thrombosis, long term anticoagulation with dicumarol is indicated.

Mechanism of cephalosporin-induced hypoprothrombinemia: relation to cephalosporin side chain, vitamin K metabolism, and vitamin K status

The mechanism of cephalosporin‐induced hypoprothrombinemia has been investigated in hospitalized patients, with respect to cephalosporin structure, vitamin K metabolism, and vitamin K status. Cephalosporins containing side chains of N‐methylthiotetrazole (latamoxef, cefmenoxime, cefoperazone, cefotetan, cefamandole) or methyl‐thiadiazole (cefazolin) all caused the transient plasma appearance of vitamin K1 2,3‐epoxide in response to a 10‐mg intravenous dose of vitamin K1, whereas two cephalosporins without a heterocyclic side chain (cefotaxime and cefoxitin) did not. The plasma accumulation of vitamin K1 2,3‐epoxide was qualitatively similar to, but quantitatively less than, that produced by the oral anticoagulant phenprocoumon. Patients eating normally had plasma vitamin K1 concentrations (176 to 1184 pg/mL) that were within the normal range (150 to 1550 pg/mL) and their clotting tests remained consistently normal for all antibiotics tested. Patients on total parenteral nutrition had lower plasma vitamin K1 concentrations (50 to 790 pg/mL) but normal clotting before starting antibiotic therapy. Of 19 parenterally fed patients, all seven treated with latamoxef developed hypoprothrombinemia, PIVKA‐II and a decrease of protein C within four days whereas 12 patients treated with cefotaxime or cefoxitin showed no clotting changes. Latamoxef‐associated hypoprothrombinemia was readily reversible by 1 mg of vitamin K1 given intravenously, but hypoprothrombinemia and sub‐normal plasma vitamin K1 could recur within two to three days. The data suggest that NMTT‐cephalosporins are inhibitors of hepatic vitamin K epoxide reductase and that a lower nutritional‐vitamin K status predisposes to hypoprothrombinemia.

Up-regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils (PMN): divergent expression in acute bacterial infections and chronic inflammatory disease

Upon cultivation with interferon‐γ (IFN‐γ ) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF) polymorphonuclear neutrophils (PMN) acquire characteristics of dendritic cells, including expression of major histocompatibility complex (MHC) class II antigens, of the co‐stimulatory antigens CD80, CD86 and of CD83, the latter considered to be specific for dendritic cells. Dendritic‐like PMN were also able to present to T cells antigens in a MHC class II‐restricted manner. To assess whether dendritic‐like PMN are also generated in vivo, cells of patients with acute bacterial infections and of patients with chronic inflammatory diseases (primary vasculitis) were tested. During acute infection up to 80% of PMN acquired CD83, but remained negative for MHC class II, CD80 or CD86. PMN of patients with primary vasculitis expressed MHC class II antigens, CD80 and CD86, but not CD83, indicating that up‐regulation of MHC class II and of CD83 are not necessarily linked to each other. Indeed, parallel studies with PMN of healthy donors showed that while IFN‐γ and granulocyte/macrophage colony stimulating factor (GM‐CSF) induced both, MHC class II and CD83, tumour necrosis factor (TNF)‐α selectively induced de novo synthesis of CD83. The function of CD83 on PMN is still elusive. A participation in the MHC class II‐restricted antigen presentation could be ruled out, consistent with the segregation of MHC class II and CD83 expression. Regardless, however, of its function, CD83 expression could serve as a marker to differentiate between acute and chronic inflammation.