Deficiency of the fifth component of complement in mice with an inherited complement defect.

The inherited complement deficiency of certain inbred strains of mice was shown to be due to an isolated lack of the fifth component of complement. The protein MuB1 (or hc'), which is present in normal mouse serum but absent from the serum of complement-deficient mice, was shown to be immunochemically related to the fifth component of human complement (C'5). C'5 hemolytic activity was specifically inhibited in human serum by mouse anti-MuB1 and in normal mouse serum by mouse antiserum to human C'5. Highly purified human C'5 reconstituted the hemolytic activity of complement-deficient mouse serum. It was, therefore, concluded that he', or MuB1, constitutes the murine analogue of the fifth component of human complement. The MuB1 concentration in normal mouse serum was found to be subject to a sex-related variation. By quantitative precipitin analysis it was demonstrated that serum from male mice contains twice as much MuB1 as that of female mice. This difference in C'5 concentration was also detected by hemolytic assay. In addition, C'6 and C'7 were also found to be subject to sex-related variations.

Occurrence of 19S and 7S anti-IgGs during hyperimmunization of rabbits with streptococci.

All 110 rabbits immunized with Group A, A-variant, and C streptococcal vaccines produced 19S anti-IgG in addition to antibodies to the streptococcal carbohydrates. 19S anti-IgG was detected by hemagglutination of rabbit red blood cells coated with rabbit anti-blood group F antibody. Antisera of 88 of these animals were also tested for 7S anti-IgG with a coprecipitation assay. This assay is based on the coprecipitation of 7S anti-IgG with complexes of streptococcal carbohydrate and anti-carbohydrate antibody. 50 of the 88 anti-Group C streptococcal antisera contained 7S anti-IgGs. In eight antisera the concentration was greater than 5 mg/ml. The data suggest a genetic influence on the occurrence of 7S anti-IgG. The eight rabbits which produced more than 5 mg/ml of 7S anti-IgG belonged to three related families. Moreover, there were families in which almost every member produced 7S anti-IgG and other families in which only 30% of the members manufactured 7S anti-IgG. The streptococcal vaccine was an especially efficient stimulus for the production of 19S anti-IgG, whereas the pneumococcal vaccine was much less effective in this respect. Furthermore, 7S anti-IgGs were not detected in antipneumococcal antisera, although the concentration of anti-capsular antibodies was similar to that of anti-carbohydrate antibodies in antistreptococcal antisera.

Bovine antigens and the formation of circulating immune complexes in selective immunoglobulin A deficiency.

We have shown that levels of circulating immune complexes are closely associated with the presence of precipitating antibodies to bovine milk proteins in individuals with selective immunoglobin (Ig)A deficiency. To test whether milk proteins are involved in immune complex formation, sera of seven IgA-deficient individuals were studied for the appearance of complexes after milk ingestion. In three of the seven, an initial fall in the level of complexes was followed by an increasing value, which peaked at 120-150 min. In another three, there was a tendency toward the formation of two peaks of complexes, the first at 30-60 min and the second at 120-150 min after drinking milk. One subject, who had had recent treatment for two separate neoplasms, had a steady level of complexes that did not change during the course of this test. After drinking milk, the molecular weight of the complexes found in the sera of one individual at the start of the milk test fell from >19S to 7-11S, and in vitro additions of progressively increasing amounts of a mixture of milk proteins or bovine gamma globulin, to sera that contained complexes produced a progressive reduction in the level of complexes detectable. We conclude that the circulating immune complexes found in some patients who lack IgA contain bovine milk proteins and that periodic fluctuation of the molecular weight of such complexes, depending upon antigen ingestion, appears likely. It remains uncertain what effect the chronic circulation of complexes has upon the clinical state of this group of patients.

Qualitative and quantitative aspects of the human antibody response to streptococcal group A carbohydrate.

The lack of impressive quantitative differences in antibody to various streptococcal extracellular and cellular antigens among patients with acute rheumatic fever, acute glomerulonephritis, and following uncomplicated streptococcal infection has prompted investigation of qualitative aspects of the antibody response among these patients. By using a radiolabeled antigenically univalent hapten derived from streptococcal A carbohydrate, affinity of serum antibody to A-carbohydrate (A-antibody) was studied by an ammonium sulfate precipitation technique. The data obtained demonstrate average association constants (K0S) of acute rheumatic fever patient sera to be significantly lower than those of acute glomerulonephritis or streptococcal infection patients (P<0.02 and P<0.001, respectively). Further analysis of the data from hapten binding studies documents the fact that the radioimmune precipitin assay for the determination of A-antibody level is little influenced by K0 but directly correlates with the concentration of antibody binding sites. These data suggest that qualitative differences in A-antibody are present between rheumatic and non-rheumatic individuals. It is unclear whether the finding of low-affinity A-antibody among acute rheumatic fever patients reflects a generalized phenomenon or one restricted to the A-antibody-A-carbohydrate system.

Hormone synthesis and secretion by rat parathyroid glands in tissue culture.

Rat parathyroid glands maintained in organ culture secrete biologically active parathyroid hormone (PTH) and synthesize and secrete labeled proteins from (3)H- or (14)C-labeled amino acids added to the medium. The amounts of biological activity and labeled protein in the medium are both inversely proportional to the calcium concentration. Some of the labeled low molecular weight protein was identified as PTH which had been synthesized and secreted in culture by preliminary isolation on Sephadex G-100 columns and further purification using an antibody to bovine PTH which cross-reacted with rat PTH. The cross-reacting antibody inhibited the biological effects of rat PTH and caused hypocalcemia in intact rats. The antibody bound some of the labeled low molecular weight protein of the medium at neutral pH so that it migrated as a large molecular weight complex on Sephadex. Biologically active, labeled PTH was recovered by dissociation of this complex in acid and rechromatography.

One antigen may form two precipitin lines and two spurs when tested with two monoclonal antibodies by gel diffusion assays.

A monoclonal antibody reactive with human immunoglobulin (Ig)G4 and a monoclonal antibody reactive with IgG1, IgG2 and IgG4 were tested with an IgG4 myeloma protein by double diffusion in a polyethylene glycol-containing gel. In a three-well Ouchterlony pattern, the IgG4 myeloma protein formed lines of double partial identity (double spur) with the two monoclonal antibodies. The two spurs lengthened and thickened with decreasing concentrations of polyethylene glycol indicating that soluble immune complexes diffused past the precipitin lines and formed the spurs. In a two-well pattern, the myeloma protein formed two lines with mixtures of the two monoclonal antibodies indicating that the immune complexes formed by the two antibodies distributed bimodally in the gel as if two types of complexes were formed. These unpredicted findings indicate that the process of antigen-antibody precipitation in gels needs to be analyzed further by using monoclonal antibodies.

Comparison of membrane-associated proteins of murine cytolytic and helper cloned T-cell lines: identification of a protein, p24, prominent in membrane fractions from cytolytic but not helper T-cells.

It has recently been reported that liposomes containing membrane components from cytolytic T-cell (TC) clones could transfer lytic activity to noncytolytic T- and B-cell lines, strongly suggesting that TC possess membrane-associated molecules which noncytolytic lymphocytes lack and which play a critical role in the lytic mechanism. It was thus of interest to compare the membrane-associated proteins from TC-lines to those of noncytolytic helper T-cell (TH) lines to determine whether any membrane-associated proteins unique to TC could be identified. Cells from three TC-lines and four TH-lines were internally labelled with [35S]methionine and then disrupted by hypotonic lysis. Low-density (plasma membrane enriched) and high-density (endoplasmic reticulum enriched) membrane fractions were isolated from each cloned cell line and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two proteins were identified which were prominent in the membrane fractions from each of the three TC-lines but not in the membrane fractions from any of the four TH-lines. One of these, p215, migrated as a broad band with an apparent mol. wt of 215,000. The other, p24, migrated as a sharp band, or tightly spaced doublet, with an apparent mol. wt of 24,000. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1 and Lyt 2 suggested that p215 was a variant of T200 found on TC-lines but not on TH-lines. Treatment of solubilized membrane proteins from TH-lines with anti-T200 precipitated a 185-kD protein seen on each of the TH-lines but on none of the TC-lines. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It therefore appears that p24 represents a previously unidentified protein which is strongly expressed by TC but not by TH and is thus deserving of further study as to its functional significance.

CLq precipitin in the sera of patients with allergic vasculitis (Gougerot-Ruiter Syndrome).

The sera from two well-documented cases of allergic vasculitis were examined for the presence of C1q precipitins. Both sera contained material capable of precipitating C1q in agarose gel. The material from one of the sera was partially purified using ammonium sulfate precipitation. Sephadex G-200 filtration, and DEAE-cellulose chromatography. Attempts to identify the nature of C1q precipitin were unsuccessful, but it resembled the high-molecular-weight precipitins of sera from patients with systemic lupus erythematosus. In a lesion, deposition of fibrinogen, immunoglobulins, and complement were noted mainly in the vessel walls. No correlation between immunoglobulin and complement deposition in the skin and the presence of C1q precipitins in the blood could be established.

Identification of immunoprecipitates in line immunoelectrophoresis.

A technique has been developed for the identification of immunoprecipitates in complex line-immunoelectrophoretic patterns. It is based on localized adsorption of individual antigens by monospecific antisera. The technique is suited for the following purposes as well: (1) testing the specificity and titer of uncharacterized antisera and (2) evaluation of binding properties of chromatographic media.

An improved method for synthesising omega-stearoyldextran for the detection of plaque forming cells specific for the alpha-(1 replaces 6) determinant.

A method for synthesising omega-stearoyldextran for the detection of plaque forming cells specific for the alpha-(1 replaces 6) determinant is described. The omega-stearoyl derivative is obtained by reacting dextran with phenylboronic acid to give a 2,4-phenylboronate, which can be subsequently esterified in a pyridine solution with stearoyl chloride. Four different preparations of omega-stearoyldextran with increasing content of stearoyl were obtained. Chemical and immunochemical properties of these compounds are given.

Detection of complement activation by counterimmunoelectrophoresis (CIE).

Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by asssaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.

High resolution isoelectric focusing of immunoprecipitated proteins under denaturing conditions. A simple analytical method applied to the study of complement component polymorphisms.

A simple analytical method for the study of structural protein polymorphisms is described. It consists of the immunoprecipitation of non-radiolabeled proteins using monospecific polyclonal antibodies followed by isoelectric focusing (IEF) under completely denaturing conditions in vertical polyacrylamide slab gels. The method uses small amounts of sample (usually unfractionated plasma or serum), requires no sophisticated equipment and allows the screening of large numbers of samples with comparatively small effort. This method has been applied in the identification of 2 human complement-component polymorphisms, C4-binding protein (C4-bp) and factor H (beta 1H).

Measurement of murine serum amyloid P component by rate nephelometry.

Murine serum amyloid P component (SAP) is an acute-phase protein that is increased 2-10-fold in concentration following appropriate inflammatory or infectious stimuli. Previous studies of the acute-phase SAP response have employed quantitative immunoelectrophoresis or radioimmunoassay to measure SAP concentration. A rate nephelometric procedure has been developed which measures SAP concentration rapidly and with equivalent or greater precision than the previously applied techniques. This simple method will facilitate experimental and clinical studies of the acute-phase response.

Non-specific precipitin reactions of IgG at low ionic strength.

Precipitin reactions apparently not involving specific antibodies were obtained in immunodiffusion experiments performed at low salt concentration between IgG and the muscle proteins, actin and tropomyosin. The precipitates, which dissolved at physiologic ionic strength, may result from electrostatic interaction of molecules of different net charge and different distribution and number of charged groups.

Detection of Candida precipitins. a comparison of double diffusion and counter immunoelectrophoresis.

The optimum conditions for detection of Candida precipitins by double diffusion and counter immunoelectrophoresis have been established. Counter immunoelectrophoresis was shown to be more sensitive than double diffusion, and its application to routine tests for Candida precipitins to cytoplasmic antigen is recommended.

Familial hypersensitivity pneumonitis.

Two families are described in which seven members of a total of 19 were found to have hypersensitivity pneumonitis due to exposure to avian antigens. Diagnosis was made on the basis of characteristic roentgenologic changes together with respiratory function and immunologic studies. The latter included screening for precipitins, macrophage migration inhibition (MMI) to specific antigens in avian serum and droppings, quantitation of immunoglobulin and alpha1 antitrypsin (AAT) levels and assessment of the complement system. Specific precipitins to pigeon and/or budgerigar serum were found in the serum of only four of the seven patients. Six of these seven patients, however, had a positive MMI. Thus, the MMI test, at least in this group of patients appeared to be a more sensitive indicator of active disease. The finding of seven members of two families with disease led to a search for predisposing factors, either genetic or environmental. Evidence for a genetic predisposition came from tissue typing studies. In the first family, both paternal haplotypes were associated with disease; the maternal haplotype HLA3,7 was not inherited by any child with disease. In the second family, the disease developed in three of four members with the haplotype HL-A2,W15, who were significantly exposed to avian antigen. In the light of recent studies showing an association between immune response (Ir) genes, histocompatibility antigens and disease susceptibility, these findings were interpreted as possible evidence for a subtle genetically linked immune defect in hypersensitivity pneumonitis. Evidence for an environmental predisposition was less clear cut, but it is interesting that members of both families used a gamma isomer of hexachlorobenzene (Nickoff) to eradicate mite infestations in their birds which might have damaged the bronchial mucosa or acted as an immunologic adjuvant in a person with underlying susceptibility to disease. The presence of subclinical respiratory disease in two family members is reported, and the importance of performing a range of investigations of respiratory function in order to detect disease and monitor its progress is emphasized.

Clinical findings in patients with SLE whose sera contain antibodies to ribosomal ribonucleoprotein.

In a clinical and serological study performed on a large series of patients with different connective tissue diseases, anti-ribosomal ribonucleoprotein (rRNP) antibodies were detected only in a small proportion of sera with systemic lupus erythematosus (SLE). SLE patients positive for anti-rRNP autoantibodies showed a significantly higher incidence of hemolytic anemia. The reasons for this surprising association are still unclear; however, this finding suggests that rRNP precipitin might be considered as a useful marker of a particular subgroup of patients with SLE.

Cell-diffusion precipitin analysis of mixed IgM-IgG cryoglobulins. An unusual kappa-chain determinant of the IgM component.

Antigenic features of monoclonal IgM kappa components of three mixed IgM-IgG cryoglobulins were studied by gel-diffusion precipitin analysis. An unusual kappa-chain determinant was revealed in the IgM components but not in other monoclonal immunoglobulins (IgM kappa, BJ kappa) or normal IgG. Antibodies to this determinant were found not only in anti-kappa, but also in anti-lambda sera, and in antisera to hidden determinants of L-chains.

Impairment of lung mucociliary clearance in pigeon fanciers.

Lung mucociliary clearance was measured in 15 pigeon fanciers. The study group was subdivided into two: a precipitin-positive group (n = 10; mean +/- SEM age 45 +/- 5 years) with circulating blood precipitins and a precipitin-negative group (n = 5; mean +/- SEM age 40 +/- 3 years) without. Clearance was measured using an objective, noninvasive radioaerosol technique. The data for both groups were compared with those of matched control groups of healthy subjects. The mean +/- SEM area under the tracheobronchial retention curves (AUC) over the 6-h observation period was 257 +/- 27 %h for the precipitin-positive group compared with 177 +/- 16 %h for its control group (p = 0.02)--a high AUC value denoting slow clearance. That for the precipitin-negative group was 282 +/- 34 %h compared with 150 +/- 15 %h for its control group (p = 0.02). Our study illustrates in pigeon fanciers involvement of the conducting airways in that a major defense mechanism of the airways--namely, mucociliary clearance--is substantially compromised. The presence or absence of precipitins appears not to be related to the degree of mucociliary clearance impairment.