S.-E. Svehag

Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma

A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.

Only amyloidogenic intermediates of transthyretin induce apoptosis

In diseases like Alzheimers disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

Passage of Undegraded Dietary Antigen into the Blood of Healthy Adults Further Characterization of the Kinetics of Uptake and the Size Distribution of the Antigen

The molecular weight distribution of ovalbumin (OA) absorbed into the blood in eight healthy adults after a test meal was analysed by high pressure liquid gel permeation chromatography (HPLC) followed by ELISA for OA. OA was found either as free OA or as aggregates of a size mainly below 700kD. The addition of OA in vitro to an antibody‐containing serum resulted in OA‐containing immune complexes (OA‐IC) of similar size. The size distribution of serum OA‐IC was more heterogeneous late than early in the 7‐h observation period. Free OA disappeared at an equal or higher rate than OA‐IC when both entities were present in the same individual. The presence of OA‐IC was related to the serum IgG antibody levels. IgG, but not IgA or IgM, could be detected in OA‐IC (in three individuals) by ELISA. The kinetics of OA appearance was followed for 48 h after a test meal in three individuals. In one person, serum OA reached peak values at 24 h and detectable amounts persisted for 48 h after the test meal. In the other test persons the OA levels peaked earlier. The present study indicates that in healthy adults dietary antigens are absorbed and circulate regularly in minute amounts apparently as native protein and/or as small immune complexes, mostly containing IgG antibodies.

Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE).

It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that normal B cells are capable of activating the alternative pathway (AP) of complement in a CR2‐dependent fashion. In this study we have investigated whether disturbances in this activity may be related to the altered phenotype of SLE B cells. Flow cytometry was used to measure expression of complement receptors and regulatory proteins on B cells from SLE patients, as well as the deposition of C3 fragments occurring in vivo or after in vitro AP activation. We have confirmed, for a proportion of the patients studied, reduced expression of CR1 and CR2 on B cells, and shown a consistency between low CR2 expression and reduced in vitro AP activation in the presence of homologous, normal serum. In addition, the B cells, like erythrocytes, bear raised levels of in vivo‐deposited C3dg, but not C3b fragments, compared with normal B cells. The erythrocytes from SLE patients were unable to inhibit in vitro AP activation by B cells in homologous serum. Finally, we demonstrated an inverse relationship between SLE disease activity index (SLEDAI) and the expression of complement receptor 2 (CR2) on SLE B cells. Thus, determination of CR2 on B cells may emerge as an additional laboratory tool in the assessment of SLE activity.

Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribution of serum antibodies to protein antigens.

The occurrence of antibodies to five dietary protein antigens in the sera from 21 healthy adults was investigated by a modified Farr assay. Antibody to ovalbumin (OA) occurred most frequently (90%) whereas only 24% had antibodies to alpha-lactalbumin (ALA). No correlation was noted between the titer of antibodies against bovine serum albumin (BSA) and OA in the single individual. The avidity constants (10(8)-10(9) l/mol) and cross-reactivities against other albumins of anti-BSA antibodies in two human sera were comparable to that of the antibodies in pooled hyperimmune rabbit antiserum. Crossed radioimmunoelectrophoresis showed serum anti-BSA and anti-OA antibodies to be predominantly of the IgG class (13/13, 10/10), occasionally of the IgA- (6/13, 1/10) and rarely of the IgM class (1/13, 0/10). Analysis by radioelectroimmunoassay (rocket immunoelectrophoresis) of the IgG subclass distribution of anti-BSA and anti-OA antibodies showed total absence of IgG3. In contrast, antibodies of the IgG4 subclass were frequently present even in sera with very low levels of total IgG4.

Passage of dietary antigens into the blood of children with coeliac disease. Quantification and size distribution of absorbed antigens.

The uptake of ovalbumin (OA) from egg and beta-lactoglobulin (BLG) from cows milk into the blood was investigated for seven hours after a test meal in five children with coeliac disease on a gluten free diet and after gluten challenge, and in five children with normal jejunal mucosa. Ovalbumin was detectable by ELISA in three of five coeliac children (maximal concentrations 8-178 ng/ml serum) and in five of five controls (maximal 4-91 ng/ml serum). Beta-lactoglobulin was detected in three of five coeliac children (maximal 0.6-6 ng/ml serum) and in two of five controls (maximal 0.5 and 50 ng/ml serum). No clear relationship was seen between maximal antigen concentrations and titres of serum IgG or IgA antibodies determined by ELISA, or as percentage antigen binding in a Farr type radioimmunoassay. Ovalbumin and beta-lactoglobulin was seen in serum of all coeliac patients and controls by HPLC fractionation in combination with ELISA, either in high MW fractions, or at the Mr of native OA and BLG, respectively. In one control degradation products (about 17 kD) of BLG were detectable in serum. The serum concentrations of OA and BLG were increased on gluten challenge in four or five coeliac children, indicating increased macromolecular passage through the gut mucosa in untreated coeliac disease.

Electron microscopy of prefibrillar structures and amyloid fibrils.

Publisher Summary Several techniques, such as X-ray crystallography, light scattering, fluorescence spectrometry, size exclusion chromatography, atomic force microscopy, and transmission electron microscopy, have been employed in studies of structural intermediates of fibril formation and fibrillar assembly of amyloid proteins. Electron microscopy, with a resolution of approximately 2 nm, offers a useful technique for the ultrastructural characterization of preprotofilaments, protofilaments, and mature fibrils formed during in vitro fibrillogenesis. For contrast enhancement of specimens, negative staining is applied. This chapter outlines the methodology used in laboratories for electron microscopic examination of negatively stained prefibrillar structures and amyloid fibrils. The A β -peptide used influences the kinetics of the fibril formation. This chapter uses A β 1–42 (Bachem, Bubendorf, Switzerland), which forms fibrils within a few hours of incubation at 37°. In order to decelerate the fibril formation enabling it to investigate early intermediates and prefibrillar structures, this chapter has performed the in vitro studies at low concentrations of A β 1–42 (170 μg/ml), as the kinetics of fibril formation is highly concentration dependent.

Soluble immune complexes in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases

The occurrence of soluble immune complexes (IC) in the cerebrospinal fluid (CSF) of 14 multiple sclerosis (MS) patients, four acute polyradiculoneuritis patients, 30 patients with other neurological diseases (OND) and 30 patients with disc prolapse (DP) was examined by a solid phase C1q‐protein A binding assay (C1q‐PABA) and a complement consumption test. IC‐positive reactions were observed only in the C1q‐PABA. The binding indices determined by the C1q‐PABA differed significantly (P < 0.01) when the MS or the OND patient groups were compared to the DP group. No significant (P < 0.1) difference was observed between the indices in the MS and OND groups. Binding indices in C1q‐PABA showed no correlation either to IgG concentration, total protein concentration or cell counts in CSF of MS patients. Three of the four polyradiculoneuritis patients were strongly IC‐positive while the fourth patient was negative. Filtration and PEG‐precipitation data indicated that a major part of the IgG‐containing IC in CSF detected by C1q‐PABA was of macromolecular nature.

Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll®)

A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.

Infants and children with cow milk allergy/intolerance. Investigation of the uptake of cow milk protein and activation of the complement system.

Seventeen children with challenge‐verified cow milk allergy/intolerance (CMAI), age 3–78 months, median 12 months, were re‐challenged with cow milk in increasing doses. All subjects developed symptoms, such as bronchospasm, rhinitis, diarrhoea, erythema or eczema. Blood samples were taken before and up to 24 h after the start of the challenge. The cow milk protein β‐lactoglobulin (BLG) was determined in serum with ELISA (lower detection limit 0.3 μg/l). BLG was detectable in five children at low levels (below 2 μg/l). Analysis of the size distribution of the BLG by size exclusion chromatography indicated immunoreactive material as small aggregates. Plasma samples were analysed by electro‐immunoassay for complement factor split product C3d, which was not demonstrable above background values in any of the cases. CMAI in infants and children may not be related to systemic activation of the complement system and may be elicited without considerable amounts of immunoreactive BLG in the circulation.