Nickolas J. Calvanico

Passive gold agglutination. An alternative to passive hemagglutination

A method is described for coating colloidal gold with protein for use in passive agglutination. Coating of the gold is quick, reproducible and the sterile filtered product is stable over a long period of storage at 4 degrees C. Colloidal gold coated with each of the 4 subclasses of human IgG was tested with rabbit, monkey and goat antisera and found to be compatible with each. The visual properties of coated gold particles resemble blood cells and appear to be equally sensitive.

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.

Immunopathology of pemphigus

The term pemphigus refers to a group of cutaneous diseases that are characterized by the development of intra-epidermal blisters and, sometimes, mucosal erosions [l] (Table 1). All forms of pemphigus are characterized by epidermal cell-cell detachment (acantholysis) which leads to (intra-epidermal) vesicle formation, and by the presence of IgG autoantibodies directed against antigenic determinants present on the cell surfaces of differentiating keratinocytes [2]. The most severe form of pemphigus is pemphigus vulgaris (PV) (Figure l), which may occur at any age, although its most common onset is in the fourth, fifth and sixth decades. PV is characterized by the presence of flaccid, exceedingly fragiIe noninflammatory bullae which usually arise on normal appearing skin. These bullae have a tendency to coalesce and rupture easily resulting in large denuded areas which are, in fact, the predominant clinical feature of this type of pemphigus. PV involves both the skin and mucous membranes and, in virtually all cases, the initial lesion affects the oral mucosa. Prior to the introduction of corticosteroids in the 195Os, PV was considered almost uniformly fatal mainly due to protein, fluid and electrolyte losses and/ or uncontrollable sepsis. Acantholysis, the histological hallmark of PV, starts with the development of epidermal intercellular edema leading to dissolution of intercellular ‘bridges’ and widening of intercellular spaces (ICS), finally ending in cell to cell detachment. These microscopic changes occur in the suprabasilar area, i.e. between the basal and spinous cell layers. Basal cells remain attached to the dermis, but are laterally detached, resembling a ‘row of tombstones’ [ 11. Endemic pemphigus foliaceus (PF) or fogo selvagem (FS) (Figure 2) is clinically and immunopathologically similar to the non-endemic form seen in other parts of the world. Clinically, PF is characterized by superficial blistering and erosive lesions that affect the skin and rarely involve mucosal surfaces [3-61. Histologically, PF is characterized by acantholysis involving the subcorneal layers of the epidermis. In

The development of granulomatous pulmonary inflammation in rabbits by aerosol challenge: I. Release of plasminogen activator by alveolar macrophages

Abstract A rabbit model of hypersensitivity pneumonitis (HP) was employed to evaluate the release of plasminogen activator (PA) as a method for monitoring the degree of pulmonary inflammation. PA release from alveolar macrophages (AM) was shown to coincide with inflammation and was maximal at approximately 2 weeks of aerosol challenge. PA release could also be induced in normal AM by peripheral lymphocytes obtained from sensitized animals after incubation with antigen. Unseparated peripheral blood mononuclear cells from experimental animals also exhibited antigen-induced PA release. These results suggest that the measurement of PA release using several different cell populations can be used to evaluate pulmonary inflammation in HP.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on IgA serum and bile levels in rats.

Serum IgA is actively transported from blood to bile against a concentration gradient in the liver by the binding of dimeric IgA to secretory component, endocytosis and transport to the bile canaliculus by vesicles. As 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to elicit hepatotoxicity, the effects of TCDD on rat serum and bile IgA levels were investigated. Rats were orally administered 50 micrograms TCDD/kg body weight in 95% corn oil: 5% acetone. At days 5, 10, 15, 20 and 30 after treatment, rats were anesthetized and a cannula inserted into the bile duct for collection of bile. In addition, blood was drawn, and, after euthanasia, the liver and thymus weights were recorded. Enzyme-linked immunosorbent assay (ELISA) techniques were employed to determine IgA in serum and bile and IgG levels in serum. Rocket immunoelectrophoresis was carried out to support ELISA results. It was found that serum IgA increased with time while serum IgG remained unchanged. In addition, while serum IgA levels were increasing, there was a concomitant decrease in biliary IgA. Thymus and liver weight changes were also observed. The data indicate that TCDD affects hepatic clearance of serum dimeric IgA and suggests that liver damage may be reflected by increased serum levels of IgA.

High temperature trypsinolysis of human IgA: Isolation of the Fcα fragment☆

Abstract The Fcα fragment of IgA1 and IgA2 was prepared by high temperature (60°C) trypsinolysis (HTT) in the presence of 1·0 M NaCl. Porcine trypsin was used in place of bovine trypsin due to its greater stability at low concentrations of Ca++ at elevated temperatures. This was necessary due to the tendency of IgA to aggregate at elevated temperatures in the presence of divalent cations. Yields of Fcα fragment varied with each IgA protein studied. The time of hydrolysis and the addition of N-ethylmaleimide (NEM) to the incubation mixture were found to influence final yields and hence the optimal conditions had to be determined for each sample. The Fcα occurred as higher polymer, dimer and monomer. The mol. wt of the monomer Fcα devoid of J chain was 58,000 which in 4 M guanidine fell to 29,000. From the size of the fragment it was determined that cleavage occurred near the end of the CH1 domain or the beginning of the hinge region. In addition to release of Fcα fragment, HTT was often accompanied by the destruction of the Fab fragment either completely or in part depending on the protein and also liberation of the covalently bound albumin and α1 antitrypsin.

γ-Glutamyl transpeptidase: Relation to immunoglobulin A and free secretory component

Abstract The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S -(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p -nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10 6 . The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.

Enzyme antigens associated with pigeon Breeder's disease. II. Isolation and characterization of acidic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (Mr=22,000). The purified trypsin had a molecular weight (Mr=25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (Mr=51,000) was shown to be a glycoprotein consisting of two polypeptides (Mr=24,000). It was readily separated from the low-molecular-weight collagenase (Mr=15,000) by gel filtration. The phospholipase (Mr=99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeders disease is discussed.