Measurement of leukocyte elastase/alpha 1-proteinase-inhibitor complex using a homogeneous and a heterogeneous enzyme-immunoassay.

The heterogeneous 2 h elastase/alpha 1-proteinase-inhibitor enzyme-linked immunosorbent assay was compared with a newly developed homogeneous 20 min immunoactivation test. The within-run and between-days coefficients of variation for both tests were satisfactory. Good agreement was found between the two tests, using specimens from 140 patients. In conclusion, the 20 min immunoactivation test is able to replace the 2 h elastase/alpha 1-proteinase-inhibitor enzyme-linked immunosorbent assay.

Measurement of proteins with the Behring Nephelometer. A multicentre evaluation.

The selective multi-protein analyser Behring Nephelometer was examined according to the ECCLS guidelines in a multicentre evaluation involving five laboratories. IgG, IgA, IgM, C-reactive protein, C3c, C4, apolipoprotein A-I and B were measured in serum, and albumin and IgG were measured in cerebrospinal fluid. All values obtained were included in the evaluation without correcting for outliers. The trial, which lasted three months and involved over 20,000 analyses, basically yielded the following results: 1. The precision was generally better than that of comparative procedures. For the majority of methods, the between-day coefficients of variation were below 4.5%. The highest coefficient of variation was 7.2% (for C-reactive protein) and the lowest 1.35% (for C3c). 2. The fraction of assigned values of control materials varied between 0.95 and 1.08. 3. Good agreement was found with results from the comparison procedures: Beckman ICS, Behring Laser Nephelometer, Hyland Laser Nephelometer. 4. No carry-over effects were observed. 5. No interferences were observed for IgG, IgA and IgM determinations using hyperbilirubinaemic or haemolytic samples. In contrast lipaemic samples and some with monoclonal immunoglobulin M showed an influence on the immunoephelometric reaction. 6. Because of the large measuring range, it is necessary to repeat analyses only in extremely rare cases. 7. During the entire evaluation period no instrument malfunctions or interruptions occurred. As a result of its reliability, the Behring Nephelometer is well suited for routine operation and emergency analyses in medium and large-sized laboratories.

Method comparison of two test kits for the determination of elastase/alpha 1-proteinase-inhibitor complex.

A method comparison of the 4 h-elastase/alpha 1-proteinase-inhibitor test kit with a newly developed 2 h-version was performed. The within-run and between-days coefficients of variation as well as the recovery of the assigned values were satisfactory for both test kits. Good agreement was found between the 2 h- and 4 h-test kits using specimens from 160 patients. In conclusion, the 2 h-test kit can be used as an alternative for the determination of elastase/alpha 1-proteinase-inhibitor complex.

Endotoxaemia in patients with Crohn's disease: a longitudinal study of elastase/alpha 1-proteinase inhibitor and Limulus-amoebocyte-lysate reactivity.

During intensive care, the severity of gut-derived systemic endotoxaemia in 16 patients with active Crohn's disease was characterized by the simultaneous determination of elastase/alpha 1-proteinase-inhibitor and Limulus-amoebocyte-lysate-reactivity. Using both assays in 15/16 patients the detectable endotoxic activity during the disease course was reflected by a parallelism between elastase/alpha 1-proteinase-inhibitor and Limulus-amoebocyte-lysate-reactivity. In 8 gut-irrigated patients, significantly lower elastase/alpha 1-proteinase-inhibitor concentrations and Limulus-amoebocyte-lysate-reactivities were measured compared with 8 gut-nonirrigated patients. The Crohn's disease activity index and the van Hees activity index as well as the inpatient time were significantly lower in lavage patients compared with gut-nonirrigated patients. It is concluded with that elastase/alpha 1-proteinase-inhibitor can be used as an alternative to the bioassayed Limulus-amoebocyte-lysate-reactivity in order to characterize systemic endotoxaemia.

Influence of 2(3-methyl-cinnamyl-hydrazono)-propionate on glucose and palmitate oxidation in human mononuclear leukocytes. Hydrazonopropionic acids, a new class of hypoglycaemic substances, VII.

2-(3-Methyl-cinnamyl-hydrozono)-propionate stimulated glucose oxidation in human mononuclear leukocytes and the stimulation was similar to that by concanavalin A. Both substances must affect glucose metabolism at two sites, the first site being before the pyruvate dehydrogenase step because of the increase of lactate plus pyruvate concentration. The second site is related to pyruvate oxidation. The hydrazone inhibited the conversion of plamitate to CO2. This effect could have caused an activation of the pyruvate dehydrogenase complex, resulting from a decrease acetyl-CoA/CoA ratio. Concanavalin A did not influence fatty acid oxidation. Both substances did not affect the CO2 formation from acetate. Mononuclear leukocytes appear to be a suitable model for the investigation of the influence of hypoglycaemic substances on glucose and fatty acid metabolism in living human cells.

Endotoxaemia in active Crohn's disease. Treatment with whole gut irrigation and 5-aminosalicylic acid.

Endotoxins in plasma were monitored during treatment in 18 patients hospitalised for acute exacerbation of Crohn's disease: systemic endotoxaemia was found on admission in all but one. The patients were randomly divided into two groups: one receiving treatment with total parenteral nutrition and steroids. To decrease the absorbable endotoxin pool, the other group was additionally treated with whole gut irrigation and 5-aminosalicylic acid was added to the lavage fluid. In most of these patients endotoxaemia cleared after intestinal lavage and they needed shorter hospitalisation. Earlier improvement was also indicated by a faster decrease of the Crohn's disease activity index and vanHees index. In the group receiving conservative treatment alone, endotoxaemia was controlled within three weeks. We conclude that endotoxaemia occurs in most patients suffering from active Crohn's disease. Control of endotoxaemia after intestinal lavage suggests that systemic endotoxaemia is caused by absorption of endotoxins from the gut. Earlier improvement after whole gut irrigation indicates its beneficial effect in active Crohn's disease.

Serum anti-lipid A antibodies in multiple myeloma and Waldenström's macroglobulinaemia.

Anti-lipid A antibodies were determined in sera from 38 patients with IgG multiple myeloma, 33 patients with IgA multiple myeloma and 38 patients with Waldenström's macroglobulinaemia using an enzyme-linked immunosorbent assay (ELISA). Compared to 34 healthy adults, significantly (p less than 0.025, p less than 0.005, p less than 0.0025) lower serum anti-lipid A antibody levels were measured for the respective patient groups. Low anti-lipid A antibody levels correlated with a higher infection rate with gram-negative bacteria in patients with monoclonal B-cell malignancies. The highest infection rate was seen in patients with simultaneous low anti-lipid A levels and secondary antibody deficiency.

Endotoxemia in intensive care patients: a longitudinal study with the limulus amebocyte lysate test.

In patients with severe underlying disease and in polytraumatized patients, clinical signs of septicemia caused by infections with gram-negative bacteria are observed postoperatively with increasing frequency. Using a photometric LAL test, a longitudinal assessment of LAL reactivity on 41 intensive care patients was performed. Postoperatively, all patients developed a septicemia of different severity with body temperatures greater than 38.5 degrees C. Dividing the individual disease course, related to body temperatures, into three phases (A-C) it was found that independent of the severity of septicemia, the majority of patients (38/41) yielded a positive LAL reactivity. In phase B (body temperature greater than 38.5 degrees C) more plasma samples contained LAL-reactive material than in phase A and C (body temperature less than 38.5 degrees C). A decline of fever (phase B to C) correlated significantly (P less than 0.05) with the change from positive to negative LAL reactivity. In patients with high leukocyte counts (15-50 X 10(9)/l) a positive LAL reactivity was found more frequently. The majority of patients (21/27) who survived were transferred with negative LAL reactivity to the general wards. The results suggest that single determinations of LAL reactivity are of limited clinical validity. Using the individual profile of LAL reactivity gained through a longitudinal assessment, data upon the development of the disease course can be obtained.

Effect of lipopolysaccharides, lipid A and interferon on the cell-mediated cytotoxicity of human leukocytes against K-562 tumor cells.

The effect of lipopolysaccharides (LPS), lipid A and interferon on cell-mediated cytotoxicity was investigated using 51Cr-labeled K-562 tumor cells as targets. As effectors, peripheral blood mononuclear cells from healthy blood donors were obtained by Ficoll gradient centrifugation: mononuclear phagocytes were eliminated by iron phagocytosis and plastic adherence. A T cell-enriched population was obtained by passing mononuclear phagocyte-depleted mononuclear cells through nylon wool columns. LPS and lipid A augmented cell-mediated cytotoxicity provided mononuclear phagocytes were present. Supernatants from LPS-treated mononuclear phagocytes and T cells enhanced mononuclear phagocyte-mediated cytotoxicity to a higher degree than LPS and lipid A alone. This finding suggests the participation of a lymphokine. In contrast, the interferon preparation increased the cell-mediated cytotoxicity both of mononuclear phagocyte-containing and of mononuclear phagocyte-depleted effectors. Here, the participation of natural killer cells as effectors is suggested.

Whole-gut irrigation as antiendotoxinaemic therapy in inflammatory bowel disease.

Marked endotoxaemia was found in 12 patients suffering from severe relapse of chronic inflammatory bowel disease. These patients did not respond satisfactorily to conservative treatment. Whole gut irrigation was employed to reduce drastically the absorbable endotoxin pool in the gut. Following the lavage the condition of all the patients thus treated improved. Plasma endotoxins dropped rapidly, serum iron increased, and in 7 febrile patients body temperature normalized. The results obtained suggest a beneficial use of whole gut irrigation as a therapeutic means in endotoxaemia associated with inflammatory bowel disease.

The polyethylene glycol precipitation technique and the particle-counting immunoassay for detection of circulating immune complex-like material in liver cirrhosis and septicemia.

Both the PEG-N technique and the PACIA were applied to determine ICLM in sera from patients with liver cirrhosis and gram-negative septicemia, that have been shown to yield positive reactions for endotoxemia with the LAL test. The precision of the PEG precipitation technique, with a stabilized fraction of human HAG of known molecular size used as a calibrator, was satisfactory. In liver cirrhosis, the detectable ICLM was mainly composed of IgG and C3, whereas in gram-negative septicemia IgM, IgG, and C3 were found. PACIA also measured significantly elevated levels of ICLM with rheumatoid factor-binding activity in both patient groups, although the two assay systems did not correlate. PEG precipitates from both patient groups analyzed by gel-chromatography and passive hemolysis test contained large immune aggregates and various amounts of immunoglobulins with specificity directed toward lipid A, the least variable portion of endotoxin. These compositional differences of detected substances may imply the presence of different types of ICLM in patients with liver cirrhosis and gram-negative septicemia.

A fully automated serum electrophoresis system.

This report summarizes an evaluation of the fully automated Olympus Hite System (AHS, Olympus, D-2000 Hamburg) for serum protein fractionation. The precision thereof exceeded that of the semi-manually processed Multiphorese System II MS 755 (Vogel, D-6300 Giessen). It proved to be rapid and easy to maintain. A comparison of the albumin values obtained by AHS and Beckman immunochemistry system showed a good correlation between both methods.

Limulus amebocyte lysate test for endotoxemia: investigations with a femtogram sensitive spectrophotometric assay.

The question of specificity of Limulus amebocyte lysate (LAL) test in the diagnosis of endotoxemia has been a limiting factor of its clinical application. Using a femtogram-sensitive spectrophotometric LAL assay 35 of 36 septic postoperative patients showed an excellent correlation (almost 100%) between positive LAL tests and culture-proven gram-negative bacteremia. Twenty patients of this group demonstrated a significant correlation (p < 0.05) between elevated total white blood cell counts and Escherichia coli Endotoxin equivalents (EcEe). All 22 liver cirrhotic patients with potentially enteric endotoxemia yielded LAL positive reactions (100%) but 9 of 22 correlated significantly as to leucocytosis and elevated EcEe (2 p < 0.01). A significant correlation between EcEe and leucocyte counts was found for all neonates and all parturients respectively (2 p < 0.001), 2 p < 0.01). In vitro tests showed that leucocytes gave positive LAL tests. Supernates of a Ficoll sedimented crude leucocyte preparations, induced LAL positivity in a dose dependent manner, while all reagents per se used in the preparation, were negative. This study suggests that heat-labile factors from leucocytes apart from endotoxin are responsible for positive LAL reactions.