Culture filtrates and total water-soluble extracts (TWSE), including ultra-sonication products of were primarily fractionated by electrophoresis in polyacrylamide gels. Other separations included chromato-graphy with Con A-Separose and human immunoglobulins from sera that contained precipitins to farmer's lung hay (FLH) antigen coupled to Sepharose 4B by the cyanogen-bromide method. Fractions were tested for their effects on the lung by the intranasal inoculation of non-immunised mice and of mice immunised with TWSE in Freund's complete adjuvant to give precipitin-negative and precipitin-positive groups. Particulate antigens of did not induce respiratory disease in non-immunised mice, but TWSE produced negligible, sporadic lesions. Precipitin-positive mice developed symptoms of extrinsic allergic alveolitis when challenged with particulate antigens consisting of living or dead spores and mycelium of or with TWSE. Elements of both Type III (Arthus) and Type IV (cell-mediated) hypersensitivities were identified histologically. The allergenicity of spores and mycelium was almost nullified by de-fatting, and activity was retained solely in a serologically inert ethanol-ether extract. Fractionation of soluble antigens produced immuno-logically-acti ve extracts, only some of which induced clinical disease. Alveolitis-inducing glycoprotein antigens obtained from Con A-Sepharose by desorption with α-methyl-D-glucopyranoside precipitated in the C region on immuno-electrophoresis. A disease-provoking group of antigens precipitating in the A region was isolated by electrophoresis in agar after primary fractionation in polyacrylamide gel. Antigens desorbed both by 3·0M acetic acid and 6·0M guanidine-HCl from immunoadsorbent columns also precipitated in the A region, but only those desorbed by guanidine-HCl induced alveolitis.

Most fractions inducing disease in precipitin-positive mice also did so in precipitin-negative, immunised animals. The formation of large hyper-sensitivity granulomata, indicative of the occurrence of a Type IV cell-mediated response, followed the introduction of bentonite particles coated with reactive fractions into the lung. Mean diameters of the granulomata did not correlate directly with the degree of alveolitis induced by the fractions. Skin reactions were bimodal and showed early Type III and late Type IV histology.


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