Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-speciuic antigen currently used for serodiagnosis ofpredinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Aralditeembedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching ofplastic sections with H202, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal anti-Netherlands Leprosy Relief Organization (NSL).

From a heterosexual male with recurrent genital herpes simplex virus (HSV-2) infection, a fresh intraepidermal vesicle on the penile skin was excised by punch biopsy, fixed and processed for electron microscopy. Differing locations and appearances of capsids and virions were studied to elucidate true host or destroyer cells. HSV-2 propagation and virion formation occurred predominantly in multi- or mononucleate spinosum cells situated at the base of the vesicle. However, some of the monocytes, young histiocytes and lymphocytic cells floating in the vesicle fluid were also involved. They harbored a small number of intranuclear capsids, designating the cells as viral (capsid) carriers. Infrequently encountered free virions in the vesicle fluid were invariably seen near neutrophils. All neutrophil granulocytes examined lacked intranuclear capsids. In contrast, distinct evidence of phagocytosis of virions and some capsids by neutrophils was found in the vesicle fluid near apical portions of spinosum cells packed with virions, or in neutrophils located between virion-loaded spinosum cells in the base lining of the vesicle. In the cytoplasm of neutrophils, single and lysosome-enclosed clusters of virions were noted. Myelin figures and vacuolation of lysosomes in free-floating neutrophils were suggestive of virion distintegration. Viral propagation and abundant virion formation, beside neutrophil and lymphocyte attack, eventually lead to spinosum cell destruction. The minimal cytopathic effects (CPE) observed in involved monocytes and lymphocytic cells floating in the vesicle fluid suggest that these cells might function as vehicles for HSV-2 (capsid) transport to the exterior or interior.

Phenolic glycolipid (PGL-I), an antigen specik to Mycubacterium leprae, was localized subcellularly in M. Zeprae residing in human skin, in M. leprae isolated from armadillo liver ('isolated M Ceprae)) and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. U l t r a c r y d o n s and Araldite sections of comparable specimens were used for comparison of localization results.A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathh sections. K4M-immunogold methods gave very satisfactory quantitive gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosectiom and the qualititatively superior Araldite sections, but new sites were also localized. Cell walls in human M. Zeprae and in isolated M. leprcre possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated A4 leprue cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprue-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. Zeprcre, and (2) htra-and extracellularly in epidermal areas where basal cells harboured M. kpme in untreated multibacillary patients.

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