N-Acetylcysteine inactivates Migration Inhibitory Factor and Delayed Hypersensitivity Reactions

In vitro studies suggest that delayed hypersensitivity follows the production of migration inhibitory factor (MIF) by sensitive lymphocytes in the presence of specific antigen. This factor arrests the migration of macrophages in vitro and in vivo. After attraction, aggregation and activation in vivo, these bystander cells produce toxic substances which induce the local reaction¹. When lymphocytes from tuberculin (PPD) sensitized guinea-pigs were incubated with PPD, cell-free supernatant fluids of the cultures contained MIF². Such migration inhibitory fluids injected intradermally with PPD, into PPD-sensitive animals, enhanced the delayed hypersensitivity reaction³. Concentrated migration inhibitory supernatant fluids injected intradermally into unsensitized animals produced local reactions of induration and erythema within 6 h; reactions reached a maximum after 16 h. Histologically there was an infiltrate of mononuclear cells at the site of injection and neutrophils and eosinophils were also present¹.     

Biochemical and Immunological Studies on Aspergillus

Two kinds of polysaccharides, glucan and glycopeptide (galactomannan-peptide), were obtained from Aspergillus fumigatus (IFO 5840) by extraction with 50% pyridine and were purified by fractional precipitation with acetone, by a column chromatography on Dowex-50 and DEAE-cellulose and by the gel filtration on Sephadex G-50 and G-200. The glycopeptide, designated APSK-66 fraction, showed both an Arthus and delayed type (tuberculin type) skin reactions in sensitized rabbits and guinea pigs. By treatment with proteolytic enzymes, the delayed type skin reactivity of APSK-66 fraction was reduced but the Arthus type skin reactivity was not affected. However, the Arthus type skin reactivity of APSK-66 fraction was completely lost by periodate oxidation, and the delayed type skin reactivity of APSK-66 fraction was retained. The APSK-66 fraction showed precipitation, complement fixation and passive hemagglutination reactions with rabbit antisera against A. fumigatus. Glucan, designated as APSK-33 fraction, did not show any immunological activity when tested in the present experiment. The chemical structures of the glucan and galactomannan were discussed.     

Studies on Continuous Enzyme Reactions

The kinetics of hydrolysis of acetyl-dl-methionine in DEAE-cellulose-aminoacylase (EC 3.5.1.14) column and DEAE-Sephadex-aminoacylase column was studied.

The rate of hydrolysis of substrate was shown to be dependent on the flow rate and independent to the dimension of the enzyme column. The rate of hydrolysis of the substrate was equal in cases of down-ward flow and of up-ward flow. The deteriorated aminoacylase columns by long period operation were reactivated by the recharge of aminoacylase to them. The continuous enzyme reaction using an aminoacylase column was superior to the batch enzyme reaction using native aminoacylase.

The enzymatic properties of the water-insoluble aminoacylase prepared by linking mold aminoacylase (EC 3.5.1.14) to DEAE-Sephadex were studied and compared with those of the native aminoacylase.

Optimum pH values for hydrolysis of several substrates by the DEAE-Sephadex-amino-acylase complex (DSA-complex) shifted about 0.5~1.5 pH units more to the acid side than those by the native enzyme. On the effects of metal ions and inhibitors, substrate specificity, optical specificity and kinetic constants, no marked difference was observed between the native enzyme and the DSA-complex. Heat stability, optimum temperature and resistance towards proteases were increased by conversion from the native form to the insoluble enzyme. It was also observed that the DSA-complex was activated by urea.     

Studies on Delayed Hypersensitivity of Antigens Isolated from Mycoplasma pneumoniae Cells

Cells of Mycoplasma pneumoniae Mac strain were fractionated into acetone-soluble and insoluble fractions. Acetone-insoluble fractions were digested with pronase and further purified by chromatography on Sephadex G-75, yielding three water-soluble fractions which were free from lipid and consisted mainly of polysaccharide-protein complex. All these water-soluble fractions possessed eliciting antigenicity to delayed hypersensitivity for M. pneumoniae as measured by skin reactions and macrophage migration inhibition tests, but not to complement-fixing antibodies. In contrast, the acetone-soluble fraction was reactive with the complement-fixing antibodies but not for the delayed hypersensitivity.     

Animal Models of Complement-Mediated Hypersensitivity Reactions to Liposomes and Other Lipid-Based Nanoparticles

Intravenous injection of some liposomal drugs, diagnostic agents, micelles and other lipid-based nanoparticles can cause acute hypersensitivity reactions (HSRs) in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement (C) system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood. These reactions can be reproduced and studied in pigs, dogs and rats, animal models which differ from each other in sensitivity and spectrum of symptoms. In the most sensitive pig model, a few miligrams of liposome (phospholipid) can cause anaphylactoid shock, characterized by pulmonary hypertension, systemic hypotension, decreased cardiac output and major cardiac arrhythmias. Pigs also display cutaneous symptoms, such as flushing and rash. The sensitivity of dogs to hemodynamic changes is close to that of pigs, but unlike pigs, dogs also react to micellar lipids (such as Cremophor EL) and their response includes pronounced blood cell and vegetative neural changes (e.g., leukopenia followed by leukocytosis, thrombocytopenia, fluid excretions). Rats are relatively insensitive inasmuch as hypotension, their most prominent response to liposomes, is induced only by one or two orders of magnitude higher phospholipid doses (based on body weight) compared to the reactogenic dose in pigs and dogs. It is suggested that the porcine and dog models are applicable for measuring and predicting the (pseudo)allergic activity of particulate “nanodrugs”.     

Immunological Studies of Human Monoamine Oxidases

Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane-bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placental monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.     

Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed-Type Hypersensitivity Reactions in Guinea Pigs

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.