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Existing anti-macrophage monoclonal antibodies are unable to differentiate between macrophages and epithelioid cells. In search of more precise reagents, we have applied recently developed antibodies to lesions of sarcoidosis and leprosy. UCHM1 and Leu-M3 stained both granulomas and surrounding histiocytes. However, in lesions with epithelioid granulomas there was a clear distinction between cells identified by RFD9 (epithelioid and giant cells) and RFD7 (macrophages in the surrounding mantle and normal tissue), whereas macrophages in the non-hypersensitivity granulomas of lepromatous leprosy were labelled by both the latter antibodies. In lung biopsies, alveolar macrophages were also labelled by both RFD7 and RFD9. These reagents may be useful for studying pathogenic mechanisms in granuloma formation.

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Full thickness skin biopsies were examined from 12 untreated leprosy patients and included five borderline tuberculoid (BT leprosy), five borderline lepromatous (BL leprosy) and two subpolar lepromatous leprosy cases. The non-lymphoid mononuclear cells present in the dermal infiltrates were analysed with immunohistological techniques using monoclonal antibodies (MoAb) which in normal tissues identify subpopulations of macrophage-like cells in tissue sections; RFD2 (recognizing all and monocytes/macrophages), RFD1 (recognizing interdigitating cells), NA1/34 (recognizing Langerhans cells) and RFD7 (recognizing only mature tissue macrophages). It was observed that using these MoAb no single cell type was unique to a particular state of the disease but that major differences in the proportions of these non-lymphoid mononuclear cells existed between BT leprosy and BL and LL leprosy. In BL leprosy lesions RFD2+ macrophages were the major cell type although a significant number (15-30%) of RFD1+ macrophage-like cells were also present. In contrast, in the dermal infiltrates of BT leprosy, RFD1+ cells were the predominant cell type (45-55%). The distribution of NA1/34+ Langerhans cells and the expression of Class II major histocompatibility (MHC) antigens was characteristically different in BT, BL and LL leprosy. The relationship between the presence and phenotype of cells considered to be involved in antigen presentation is discussed in relationship to the different clinical states in leprosy.

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A method is described which can be used to quantitate class II MHC antigens (HLA-DR) expressed by cells within tissue sections. A mouse anti-human HLA-DR monoclonal antibody is directly conjugated to the fungal enzyme glucose oxidase. The enzyme, in the presence of its substrate can be used to reduce tetrazolium salts to insoluble coloured formazans. The coloured reaction product is proportional to the amount of antigen and can be eluted from cells and measured spectrophotometrically. The application of this technique to a study of the expression of HLA-DR antigens, functionally significant molecules, by mononuclear cells in the cutaneous lesions of leprosy, is described. When a quantitative measure of the HLA-DR expression was related to the area of the granulomata, significant differences in the HLA-DR expression by cells in the infiltrates associated with lesions of tuberculoid and lepromatous leprosy were observed.

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The virulence of Bordetella bronchiseptica in gnotobiotic piglets was studied by intranasal infection with 11 cultures derived from eight strains isolated from pigs (4), dogs (2), a human subject and a monkey. Six of the cultures contained organisms in phase I and five contained phenotypically different phase-III or -IV organisms. Of the phase-III and -IV cultures, four were derived from strains that had been isolated in phase I. Colonisation of the nasal cavity was investigated by counting bacteria in nasal swabs and washings. The toxigenicity of cell extracts from each strain and variant was determined by tests of lethality in mice or of cytopathogenicity in cell cultures. The results showed that two phase-I cultures from pigs colonised the nasal cavity and respiratory tract of gnotobiotic piglets better than did four phase-I cultures from other species. Phase-I organisms invariably produced capsules, fimbriae and mannose-resistant haemagglutination of guinea-pig erythrocytes. Four of five cultures in phases III and IV consisted of organisms that did not produce capsules, fimbriae or haemagglutination and colonised the nasal cavity poorly. Phase variation from I to III occurred in culture and in vivo, but variation from III to I occurred in vivo only and was accompanied by enhanced colonisation. Gnotobiotic piglets infected with porcine phase-I organisms exhibited atrophy of the nasal turbinate bones after 28 days; these organisms produced significantly more toxin than did bacteria in phase I from other species, or those in phases III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

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This paper describes a method for processing fresh tissue that allows immunohistological analysis on paraffin sections. The method is based on the use of periodate-lysine-paraformaldehyde fixation. The effects of variation in fixation time, concentration of paraformaldehyde, dehydration, clearing, wax embedding and enzyme treatment of cut sections were examined. An optimal processing procedure was established that retains good tissue morphology and allowed 21 out of 27 monoclonal antibodies tested to be used successfully on paraffin sections to identify all major cell subpopulations by their membrane antigenic characteristics. The value of this approach in studying the immunopathology of potentially dangerous infectious diseases and in leukaemia/lymphoma diagnosis is discussed.

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Full thickness skin biopsies from four patients with borderline lepromatous leprosy (BL leprosy) have been examined. Immunohistological techniques have been employed to analyse the non-lymphoid mononuclear cells present in the dermal infiltrates associated with the BL lesions. This analysis was performed using three monoclonal antibodies, RFD2 (recognizing macrophages), RFD1 (recognizing interdigitating cells) and NA1/34 (recognizing Langerhans cells). It was found that the vast majority of non-lymphoid mononuclear cells in the lesions were RFD2+ macrophages. However, a significant number (15-30%) of macrophage like cells were RFD1+ interdigitating cells. A very small number of NA1/34+ Langerhans cells were also identified within the dermal infiltrates. Combination immunohistology and Ziehl Neelsen staining revealed that all these cell types could be found containing the Mycobacterium leprae organisms. The proportions of parasitized cells within each subpopulation was equivalent to the overall proportion of each cell type within the infiltrate. The significance of parasitism of cell types thought to be involved in antigen presentation and induction of immune responses is discussed.

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