Reactive macrophage activation syndrome: a simple screening strategy and its potential in early treatment initiation.

QUESTIONS UNDER STUDY: starting treatment of reactive macrophage activation syndromes as early as possible (rMAS, haemophagocytic lymphohistiocytosis), e.g., with intravenous immunoglobulins (IVIG), seems to be essential for optimal outcome. However, there is no diagnostic gold standard which reliably indicates need for early treatment. We used a simple screening strategy consisting of serum ferritin measurements and/or morphological assessment of haemophagocytosis and compared the studied patient population with published series. METHODS: Retrospective analysis of clinical and laboratory data of 57 patients experiencing 60 episodes of rMAS. RESULTS: Screening by serum ferritin measurements and/or morphological assessment of haemophagocytosis of patients presenting with a systemic inflammatory response syndrome (SIRS) indicates that rMAS might be considerably more frequent than stated in the literature. Serum ferritin exceeded >10,000 microg/L in 91% rMAS episodes. Although the patient population studied was otherwise similar in most aspects to the published rMAS series, the fact that 40% of patients fulfilled the criteria for Still's disease (SD) as the disorder underlying rMAS is remarkable and questions the distinct nature of the two diseases. IVIG responders and non-responders did not differ regarding their initial characteristics with exception to the timepoint of IVIG administration, confirming the importance of early treatment initiation. Malignancy-associated rMAS however, has a poor prognosis and seems to be refractory to manipulation with IVIG in most instances, even when responding initially. CONCLUSIONS: rMAS has to be considered in patients with a SIRS- or SD-like clinical presentation. Hyperferritinaemia >or=10,000 microg/l seems to be a good marker for defining patients with or at risk for developing rMAS and should be completed with a morphological assessment of haemophagocytosis. The perception of acute SD and rMAS as two distinct entities has to be questioned at least in a subgroup of patients.

Hyperferritinemia as indicator for intravenous immunoglobulin treatment in reactive macrophage activation syndromes.

The underlying mechanisms of reactive macrophage activation syndromes (rMAS) are not understood in detail, and there is no specific treatment. This observational study was prompted by intravenous immunoglobulin (IVIG), dramatically halting two distinct rMAS episodes in the same patient. We evaluated the potential benefits of IVIG administration in treating fulminant rMAS and the usefulness of monitoring serum ferritin levels as an indication for emergency treatment with IVIG. Ten females and 10 males experiencing 22 episodes of rMAS were recruited on the basis of serum ferritin levels >or=10,000 microg/l and/or direct evidence of haemophagocytosis in 11 intensive care units in secondary and tertiary care hospitals in Switzerland between October 1993 and May 2000. In individual patients, serially measured ferritin was closely related to disease activity. Abrupt increases of up to >100,000 microg/l could be observed within hours. Rapid and profound beneficial effects of emergency IVIG treatment were seen in 12 episodes of rMAS accompanied by a prompt decrease of serum ferritin. IVIG produced partial or delayed improvements in 5 patients. No apparent effects were seen in 5 patients. IVIG was only successful if started early during the ferritin run-up to peak values. In conclusion, IVIG is effective in at least a subgroup of adult rMAS when started at the beginning of the macrophage activation process. The monitoring of serum ferritin levels might be helpful in detecting macrophage activation in order to commence IVIG treatment early enough.

Elevation of soluble Fas and soluble Fas ligand in reactive macrophage activation syndromes.

Derailed T-cell activation can give rise to life-threatening macrophage activation, the final common pathway of the different forms of reactive macrophage activation syndromes (rMAS). Besides inappropriate activation of the immune system, impaired termination of immune responses might be another mechanism leading to rMAS. The Fas (CD95)/Fas ligand (CD95 ligand) system functions in turning off immune responses by executing activation-induced cell death (AICD). Soluble Fas (sFas) and Fas ligand (sFasL) can interfere with their corresponding membrane-bound counterparts, qualifying them as potential parameters of impaired immune termination. Hence, sFas and sFasL were analyzed in sera of rMAS patients. We show that soluble Fas/CD95 (sFas) is elevated >2 SD over the mean of controls in all 8 rMAS episodes studied (mean 12.08 +/- 6.12 ng/mL, range 3.7-20.2; controls 2.46 +/- 0.49, range 1.5-2.9). sFasL was detected during five rMAS episodes (0.70 +/- 0.49 ng/mL, range 0.16-1.28; controls all below the limit of detection of 0.1). In addition, both parameters decrease during convalescence, reflecting clinical evolution. In conclusion, sFas seems to be consistently elevated during acute rMAS. sFasL is detected only in a subgroup of our adult rMAS patients extending the recent finding of sFasL elevation in a majority of children with macrophage activation syndromes (Hasegawa et al. Blood 1998;91(8):2793-2799). By interfering with AICD, sFas and sFasL might contribute to the pathogenesis of at least a subset of rMAS.

Effects of unprocessed and processed cardiopulmonary bypass blood retransfused into patients after cardiac surgery.

BACKGROUND: The aim of this prospective study was to compare the effect of autologous unprocessed to processed residual cardiopulmonary bypass blood (CPB) on patients' laboratory and clinical parameters and outcome. METHODS: 20 patients undergoing elective coronary artery bypass surgery were randomized to receive either unprocessed CPB blood (control group) or processed CPB blood employing the Continuous AutoTransfusion System (CATS; Fresenius, Bad Homburg, Germany). We have shown that this method eliminated >93% of activated mediators. Serial laboratory parameters including complement activation, coagulation factors and the stimulation of IL-6 and IL-8 were compared with clinical side effects and patients' outcome. RESULTS: Compared to control patients, retransfusion of unprocessed CBP blood significantly increased heparin, free plasma hemoglobin and D-Dimers. Postoperatively, three patients in the control group and two patients in the CATS group required prolonged mechanical ventilation or developed infections associated respectively with elevated C3a (desArg) or IL-6 concentration. CONCLUSIONS: CATS-processing of CPB blood provided a high-quality red blood cell concentrate, resulting in a reduced load of retransfused activated mediators.

Immunological effects of therapeutic immunoadsorption with respect to biocompatibility.

The activation of the complement system leading to generation of anaphylatoxins and the membrane attack complex depends on the chemical nature of the adsorptive system and the anticoagulation used. The method of the primary separation determines the presence of cell debris in the plasma as well as the extent of platelet activation. The particular role of anticoagulation and its properties to prevent/reduce complement activation on immunadsorption material is discussed and the combined use of citrate and heparin is proposed. The quality of the reinfused plasma--as discussed on the example of LDL-apheresis--is therefore influenced by the amount of the activated split products. This determines finally the extent of cellular activation during therapeutic immunadsorption when receptor-dependent activation of cells by C3a(desarg) and C5a(desarg) can occur.

Qualitative assessment of blood washing with the continuous autologous transfusion system (CATS)

A number of different blood-processing methods can be used at the end of cardiopulmonary bypass (CPB) to improve the quality of autologous blood. They include centrifugation, hemofiltration and cell-washing. They differ in processing time required, cost of disposables and the quality of the processed autologous blood product. The newly developed continuous auto-transfusion system (CATS: Fresenius AG, Bad Homburg) uses a continuous cell-washing method. In a prospective study, the oxygenator blood of 10 patients was processed at the end of cardiac surgery with CATS and the quality of autologous blood before and after processing was compared. The processing volumes and the time required were recorded. The concentrations and elimination rates of blood parameters and waste products such as activated coagulation and complement products were measured. At the end of CPB a mean volume of 1,010 +/- 174 ml diluted oxygenator blood was processed and concentrated to 310 +/- 88 ml in 11.0 +/- 2.2 mins. Cellular elements such as erythrocytes and leucocytes were mostly retained and their concentration showed a significant increase after processing (250% and 210% respectively; p < 0.01). Thus, the blood processing with CATS resulted in an excellent hemoconcentration (hematocrit 62 +/- 3 vs. 24 +/- 4% before processing) with a consistent reproducibility. On the other hand, the CATS concentrate showed a significant loss of autologous plasma proteins. Likewise, all water soluble elements such as waste products are significantly lower in concentration after processing and, if calculated by quantity, they show a high elimination rate (> 93%). In conclusion, the continuous autologous transfusion system permits an automated, rapid and continuous processing of autologous blood yielding a standardised high quality erythrocyte concentrate.

Hereditary angioedema: uncomplicated maxillofacial surgery using short-term C1 inhibitor replacement therapy.

We report on the successful use of a pasteurized C1 inhibitor (C1-INH) concentrate during dental surgery of a patient affected by hereditary angioedema. The patient recovered fully without complications or side effects. Within 30 min, the first 1,000 U of C1-INH concentrate raised C1-INH concentration from 19 to 55% and function from 40 to 90% of the normal mean. When measured 4 h after the second injection, a further increase of the C1-INH concentration to 86% and a function of 106% relative to the normal mean was observed. Within 2 weeks C1-INH concentration returned to pretreatment level, while the function remained above this value. Serum liver enzyme values did not change and no anti-C1-INH alloantibodies were detected 10 months post-replacement therapy. We conclude that in patients affected by C1-INH deficiency, dental surgery and likely other traumatic procedures can be safely performed in conjunction with C1-INH replacement therapy even without preliminary treatment.

Low number of complement C3b/C4b receptors (CR1) on erythrocytes from patients with essential mixed cryoglobulinemia, systemic lupus erythematosus and rheumatoid arthritis: relationship with disease activity, anticardiolipin antibodies, complement activation and therapy.

Our aim was to assess whether the amount of complement C3b/C4b receptors (CR1) on erythrocytes shows a correlation to disease activity in various connective tissue diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and essential mixed cryoglobulinemia (EMC). Using an anti-CR1 monoclonal antibody, 26 patients with SLE, 34 with RA and 22 patients with EMC were investigated for erythrocyte CR1 expression. The control group consisted of 30 healthy individuals. The mean number of CR1/erythrocyte in the control group was 568 +/- 197 (range 174-1060), significantly higher than studied (EMC:379 +/- 248; p = 0.0005;SLE 147 +/- 56, p less than 0.0001; RA 298 +/- 177, p less than 0.0001). In patients with RA and in SLE, but not in patients with EMC, the number of CR1 numbers and anticardiolipin antibody (aCl) titers (r2 = 0.493; p = 0.034). A statistically significant correlation between CR1 numbers and CH50 values was found in patients with SLE, while in 3 patients with RA 4 months of therapy with cyclosporine A led to a further 30% reduction in CR1 number. Our conclusions are that (a) the decreased expression of erythrocyte CR1 is apparently a common feature of patients with various connective tissue diseases; (b) several acquired factors such as disease activity, complement activation, aCl and drugs may contribute to the loss of CR1 from erythrocytes; (c) in patients with RA and SLE, but not in patients with EMC, CR1 enumeration on erythrocytes may serve as a variable for clinical monitoring.

Use of high-dose intravenous immunoglobulin in systemic lupus erythematosus: report of three cases.

Three patients with life-threatening manifestations of systemic lupus erythematosus (SLE), unresponsive to conventional high-dose corticosteroid and/or immunosuppressive therapy were treated with intravenous polyspecific IgG (IVIG). Following IVIG infusion, lupus encephalitis in the first patient quickly resolved and the impressive improvement of the clinical status was associated with a transient increase in C1q-binding activity. The daily infusion of IgG had to be suspended after three days in the second patient with encephalitis and nephritis, because the renal function rapidly deteriorated; subsequently, six plasma exchanges led to an almost complete recovery. Finally, leukocyte and platelet counts increased and remained within normal range following IgG therapy in the third patient having SLE-associated leuko- and thrombocytopenia. In all three patients a decrease in anti-DNA antibody levels and an increase in total complement hemolytic activity were detected after therapy.

[Serum immunoglobulin concentrations in patients following bone marrow transplantation and preventive administration of high-dose nonspecific or normal-dose CMV-specific immunoglobulin]. / Serumimmunglobulinkonzentrationen in Patienten nach Knochenmarktransplantation und prophylaktischer Gabe von hoch-dosiert unspezifischem oder normal-dosiert CMV-spezifischem Immunglobulin.

Ten consecutive bone marrow transplant recipients were assigned alternatively to receive either a high dose polyspecific intravenous immunglobulin (HDIVIG) 0.5 g/kg weekly X 14, or a conventional dose of a CMV specific intravenous immunglobulin (CMVIG) 0.1 g/kg every three weeks. Before and after each application we determined the levels of total IgG, IgG subclasses, IgG 1, IgG 2, IgG 3, IgG 4, total IgA and IgM and of specific IgG and IgM antibody concentrations against endotoxin, lipid A, streptococcus A, hemophilus, EBV and CMV. In the HDIVIG, total IgG rose from 11 (median, range 2-11) g/l to 25 (22-32) g/l by day 89, in the CMVIG group, IgG dropped from 11 (10-15) g/l to 9 (6-12) g/l. In the same period the mean levels of IgG subclasses expressed as % of normal plasma pool increased in the HDIVIG group in all subclasses, not in the CMVIG group. The differences were significant, however, only for IgG 1 and IgG 2. IgG-antibodies against CMV, EBV, endotoxin, streptococcus and hemophilus did increase significantly in the HDIVIG group, but not in the CMVIG group. No increase was seen in both groups in lipid A antibodies and in specific IgM antibodies. Each application of CMVIG induced a transient increase of anti-CMV antibodies. We conclude that HDIVIG induces reproducibly an increase of specific and non-specific IgG antibodies. No attempt was made to assess clinical outcome. However, these results provide a rational basis for further therapeutic studies.

Quantitative assessment of human neutrophil chemiluminescence induced by opsonized Escherichia coli K-12.

The interaction of opsonized E. coli K-12 bacteria and polymorphonuclear leukocytes (PMN) was quantified, using luminol-enhanced chemiluminescence (CL) as a parameter of PMN stimulation. On a double-logarithmic scale light emission depended on the opsonin concentration used during pre-opsonisation. The most potent CL-inducing agent was fresh human serum, and its stimulatory activity depended on an intact complement (C) system. Both inactivation of C by heating or blocking the classical C pathway with EGTD decreased the CL-inducing potency by a factor of 8 to 16. Hypogammaglobulinemic heated serum mediated little CL. IgG for intravenous use mediated CL generation, but reduction/alkylation and sulphitolysis reduced the stimulatory power. Evidence is presented that the anti-K-12 antibodies within commercial IgG and IgM used for substitution do not improve the stimulatory power of IgG-deficient, IgM- and C-sufficient serum, unless very high Ig concentrations are substituted.

Tetanus toxoid-anti-tetanus toxoid complexes: a potential model to study the complement transport system for immune complex in humans.

Complement and its receptor on erythrocytes appears to play a physiological role in the elimination of large immune complexes (IC) in monkeys, and a similar system is likely to work in humans. Here we define a safe IC model which is suitable for clinical investigations. Soluble tetanus toxoid (TT)-human anti-TT (IgG) antibody complexes were prepared in large antibody excess. The size of the complexes was approximately 45 S. When incubated in normal human serum, 50% of the IC increased further in size, but remained soluble, and bound rapidly to human erythrocytes in vitro. This binding was shown to require intact classical pathway function. When injected into normal guinea-pigs a comparable proportion of IC bound immediately to blood cells (mainly to platelets). No platelet binding of IC occurred in C4-deficient guinea-pigs, but this binding was restored when C4 was supplied. Initial immune complex elimination was faster in C4 deficient than in C4-supplemented and normal guinea pigs. Thus classical pathway function appeared to be necessary for the normal processing, transport and elimination of TT-anti-TT complexes.

Modulation of human monocyte Fc receptor function by surface-adsorbed IgG.

Fc receptor-mediated phagocytosis was measured with monocytes subjected to various treatments. Monocytes exposed to IgG during their adherence, or after they had adhered to a surface, experienced functional impairment. This was manifested in the requirement of a higher antibody density on target particle for efficient phagocytosis, and in an enhanced susceptibility to inhibition by fluid-phase IgG. The impairment was found to be due to an interaction of IgG adhering to the surface with the Fc receptors. This effect could be induced with monomeric IgG, devoid of IgG aggregates or immune complexes. IgG coatings that resulted in inefficient Clq fixation promoted considerable functional impairment of monocytes within 1 hr. In addition, the prolonged contact of monocytes with polystyrene in the absence of IgG also led to a functional reduction. The study points to a compromised function of phagocytes exposed to artificial surfaces.

Hemolytic activity of leukemic sera: the role of complement and sucrose.

In sera of patients with acute myeloblastic leukemia (AML), hemolytic activity can be demonstrated in vitro in the presence of sucrose. To investigate the nature and the mode of action of this hemolytic activity, serum samples from 24 patients with AML were studied by incubation of normal human erythrocytes together with patient serum in the presence of sucrose at low ionic strength (inverse sucrose hemolysis test, ISHT). Fifty-five percent of the serum samples collected during the active stage of the disease gave a hemolysis rate of greater than 4%, whereas in remission only 15% of the samples lysed erythrocytes. Substitution of raffinose, lactose, or polyethylene glycol 400 for sucrose resulted in an almost complete failure of hemolysis under standard conditions, indicating a minor role of the low ionic strength in the ISHT. Heat inactivation, preincubation with inulin, and addition of EDTA, Mg2+-EGTA, or heparin completely abolished hemolytic activities of AML sera when the incubation was carried out for 30 minutes (standard conditions of the ISHT). A prolongation of the incubation time resulted in delayed hemolysis only with the Mg2+-EGTA-treated AML sera. The kinetics of this hemolysis by Mg2+-EGTA-treated AML sera were similar to those of normal human serum in the presence or absence of Mg2+-EGTA. Hemolysis was also obtained by performing the ISHT with normal sera and erythrocytes preincubated with AML sera. These observations suggest a mediation of membrane modification of normal human erythrocytes by AML sera in the presence of sucrose, resulting in an activation of the classical pathway of complement.

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

An immunochemical study of the synovial fluid can contribute to the distinction between rheumatoid and non rheumatoid arthritis.

Synovial fluids from 102 patients affected by various joint diseases have been analyzed for their protein, immunoglobulin and beta 2-microglobulin contents, for the total and alternative pathway hemolytic activities of the complement components, for the presence of rheumatoid factors and Clq binding materials. The aim of this study was to verify whether an immunochemical analysis of the synovial fluid could help to distinguish rheumatoid arthritis from other arthropathies. With regard to this, we emphasize that the latex test, the Clq binding assay, as well as the measurement of C3d and beta 2-microglobulin concentrations in synovial fluids, were the most helpful assays. An immunochemical synovial score was calculated summing the results of these four tests, thus making the recognition of rheumatoid arthritis, among the various inflammatory joint diseases, easier.

An extended C1q-binding assay using lactoperoxidase- and chloramine-T-iodinated C1q. Immediate distinction between immune-aggregate-mediated and non-immune-aggregate-mediated C1q binding.

An extension of the C1q-binding assay for the detection of immune-aggregate-mediated and non-immune-aggregate-mediated C1q binding is reported. The assay involves the use of two different C1q preparations, one radioiodinated by means of lactoperoxidase (LPO-125I-C1q) and the other by means of chloramine-T (CT-125I-C1q). The treatment with CT for 20 min at room temperature before iodination for 1 min led to abolishment of the C1q-binding capacities to complexed IgG: approximately 50% of LPO-125I-C1q but only 2% of CT-125I-C1q bound to 80 micrograms/ml of IgG forming part of tetanus toxoid/anti-tetanus toxoid complexes or to 200 micrograms/ml of heat-aggregated human gamma globulin. Similar results were obtained with staphylococcal protein-A-aggregated IgG. CT-treated C1q was haemolytically inactive. In contrast to the results with complexed IgG, CT treatment did not markedly reduce binding capacities of C1q to heparin: approximately 55% of LPO- and CT-125I-C1q were bound by 127 U/ml of commercial heparin in normal human serum. Both C1q preparations bound to a comparable extent to fibronectin, fibrinogen, and various bacterial endotoxins. When the LPO- and CT-125I-C1q-binding patterns obtained on serum samples from patients with systemic lupus erythematosus, rheumatoid arthritis, or essential mixed cryoglobulinaemia were compared with binding patterns observed using laboratory reactants, an immediate detection of non-immune-aggregate-mediated C1q binding became possible.

Symmetrical reactive oligoarthritis after Campylobacter jejuni enteritis--case report and study of the synovial complement.

A 34-year-old man, in whom sacroiliitis had been diagnosed 5 years previously, presented in July 1982 with reactive arthritis following Campylobacter jejuni enteritis. Diarrhoea was stopped by erythromycin but joint effusion recurred. In order to clarify the relationship between Campylobacter jejuni and the immunological system, we proceeded with a study of the synovial complement. The results were compared with those obtained in some other arthropathies.