Robert G. Garrison

Pharmacological Differentiation of Allergic and Classically Conditioned Asthma in the Guinea Pig

A whole-body plethysmographic technique was developed and then used to detect experimentally induced asthma in guinea pigs and to assess pharmacological treatments of allergic and classically conditioned attacks. Inhalation of a beta adrenergic compound (isoproterenol) controlled both forms of attack. Atropine and methscopolamine, parasympathetic blocking agents, prevented conditional but not allergic attacks; diphenhydramine, an antihistamine, prevented allergic attacks; and methysergide, which blocks serotonin (which is believed to trigger human asthma), prevented neither. The guinea pigs allergic reaction is probably the result of a bronchospasm induced by histamine released in tissue of the airway by a local combination of allergen and antibody. The conditional attack is believed to be a constriction of the airway mediated by parasympathetic fibers of central origin.

Intrahyphal hyphae in corneal tissue from a case of keratitis due to Lasiodioplodia theobromae

Lasiodiplodia theobromae, a recognized plant pathogen, was isolated in culture from a case of human mycotic keratitis. Chemotherapy with a variety of azoles was unsuccessful and the lesion was removed surgically. Electron microscopy of thin sections of the excised corneal tissue revealed several examples of intrahyphal hyphae, a unique process described previously in in vitro cultures of various zoopathogenic fungi. We believe this to be the first report of the presence of intrahyphal hyphae in parasitized animal or human tissue. The demonstration of this process in vivo is thought to be consistent with the hypothesis that intrahyphal hyphae might represent an attempt by the invading fungus to survive in an otherwise unfavourable environment.

Spontaneous feline sporotrichosis: A fine structural study

Fine structural details of the parasitic yeastlike phase of Sporothrix schenckii contained in biopsy tissue from a naturally-occurring case of disseminated feline sporotrichosis are described and illustrated by electron microscopy. Both free and phagocytosed fungal cells were observed. The fungal cells were contained within an extracellular, electron transparent vacuolar area which was bounded by a limiting membrane of probable host origin. The yeastlike cells were characterized by a conspicuous layer of osmiophilic microfilaments which occurred along the outermost surface of the cell wall. In many yeastlike cells, scattered, membranebound vacuoles containing electron opaque material were observed in the cytoplasm. Asteroid bodies were not observed.

Role of the conidium in dimorphism of Blastomyces dermatitidis

Fine details of yeastlike cell development of Blastomyces dermatitidis from its conidium are described and illustrated by electron micrographs. When cultured in an enriched medium at 37 °C, conidia of two strains of B. dermatitidis readily underwent ultrastructural changes consistent with mycelial to yeast dimorphism. Although hyphal cells contained in the conversion cultures were observed consistently to undergo profound degenerative changes, the conidia rapidly germinated to give rise to short germ tubes which subsequently enlarged to form intermediate yeast mother cells (YMC). The wall of the germ tube arose from the innermost layer of the wall of the germinant. During the transition globoid osmiophilic inclusions of unknown origin and function were observed in vacuolated areas of the germ tube and YMC cytoplasm. Yeastlike daughter cells then budded from the intermediate YMC. Since transformation was readily accomplished under in vitro conditions favoring mycelial to yeast dimorphism, it is suggested that the conidium of B. dermatitidis represents the primary infective unit of this pathogenic fungus.

The possible role of uric acid in the ecology of Histoplasma capsulatum

The results of this study indicate thatHistoplasma capsulatum in its saprophytic form is able to utilize the major nitrogenous constituent of avian manure as a nitrogen source. In addition, the enzymes responsible for the pathway of uric acid degradation to inorganic nitrogen have been demonstrated in cell-free systems. These enzymes include uricase, allantoinase, allantoicase, and urease. The uricase ofHistoplasma appears to be a cell wall or cell membrane-associated enzyme, while the other enzymes were located in the soluble portion of cell-free extracts. Cell-free extracts ofCryptococcus neoformans are actively uricolytic. It is suggested that this ability ofH. capsulatum hyphae to utilize uric acid and related compounds as growth substrates may in part explain the indisputable ecologic association of this pathogenic fungus with avian and possibly chiropteran-associated soils and habitats in those areas endemic for histoplasmosis.The results of this study indicate thatHistoplasma capsulatum in its saprophytic form is able to utilize the major nitrogenous constituent of avian manure as a nitrogen source. In addition, the enzymes responsible for the pathway of uric acid degradation to inorganic nitrogen have been demonstrated in cell-free systems. These enzymes include uricase, allantoinase, allantoicase, and urease. The uricase ofHistoplasma appears to be a cell wall or cell membrane-associated enzyme, while the other enzymes were located in the soluble portion of cell-free extracts. Cell-free extracts ofCryptococcus neoformans are actively uricolytic. It is suggested that this ability ofH. capsulatum hyphae to utilize uric acid and related compounds as growth substrates may in part explain the indisputable ecologic association of this pathogenic fungus with avian and possibly chiropteran-associated soils and habitats in those areas endemic for histoplasmosis.

The uptake of low molecular weight sulfur-containing compounds by Histoplasma capsulatum and related dimorphic fungi

A number of low molecular weight organic sulfur-containing compounds were tested for their effect on the respiratory activity of yeastlike and mycelialH. capsulatum. Of the compounds tested, L-cyst(e)ine was found to give maximum stimulatory effect on yeastlike phase respiration. The D- and meso isomers of cyst(e)ine as well as substituted derivatives were much less effective in the stimulation of respiratory activity of yeastlikeH. capsulatum. Respiration of homologous mycelial phase cell suspensions was depressed in the presence of L-cystine as substrate, while respiratory activity of yeastlikeB. dermatitidis andS. schenckii was unaffected. Whole cell suspensions of yeastlikeH. capsulatum actively transported S35-labeled L-cystine and methionine but apparently not β-mercaptoacetate-S35. Mycelial phaseH. capsulatum and the yeastlike and mycelial phases ofB. dermatitidis andS. schenckii were observed to take up S35-labeled L-cystine to a much lesser degree than yeastlikeH. capsulatum as determined on a dry weight basis. These results suggest significant differences in the transport and subsequent intracellular mechanisms of metabolism of low molecular weight sulfur-containing α-amino acids and related compounds by yeastlikeH. capsulatum and its corresponding mycelial phase as well as the dimorphic fungiB. dermatitidis andS. schenckii. Eine Anzahl organischer, Sulfur-enthaltender Verbindungen mit niedrigem Molekulargewicht sind betreffs ihrer Wirkung an der Atmungsaktivität von hefeähnlichem und myzelialemH. capsulatum untersucht worden. Von den untersuchten Verbindungen gab L-cyst(e)ine die größte Reizwirkung an der Atmung der Hefephase. Die D- und Meso-Isomers von Cyst(e)ine so wie auch die substituierten Derivatives waren in der Reizung der Atmungsaktivität der Hefephase vonH. capsulatum weniger wirksam. Die Atmung der Suspension von Zellen der homologen Myzelphase war in der Gegenwart von L-cystine als Substrat unterdrückt, während die Atmungsaktivität der Hefephase desB. dermatitidis und die desS. schenckii unbeeinflußt blieb. Suspensionen von ganzen Zellen der Hefephase desH. capsulatum transportierten wirksam S35 L-cystine und Methionine, aber anscheinend nicht beta-mercaptoacetate-S35. Myzelphase-H. capsulatum und Hefeund Myzelphasen desB. dermatitidis undS. schenckii nehmen S35-L-Cystine zu einem geringeren Grade auf denn Hefephase-H. capsulatum wie es am Trockengewicht festgestellt worden ist. Diese Ergebnisse legen es nahe, daß wesentliche Unterschiede im Transport und in dem nachfolgenden Intracellularmechanismus des Stoffwechsels von den Sulfurenthaltenden alfa-Aminosäuren mit niedrigem Molekulargewicht und verwandten Verbindingen durch die Hefephase desH. capsulatum und der bezüglichen Myzelphase, so wie auch durch die Doppelphasenpilze:B. dermatitidis undS. schenckii bestehen.

Electron cytochemical demonstration of the capsule of yeast-like Sporothrix schenckii

Staining of yeast-like cells of Sporothrix schenckii by the cationic dye Alcian blue 8GX and lanthanum nitrate permitted the demonstration in thin sections of an electron opaque material localized at and along the outermost wall surface. This material was associated with the characteristic microfibrillar layer of the external cell wall. It was absent or largely removed when cells were grown in shaken liquid culture or after washing of cells from static cultures. It is believed that this loosely-bound material represents a capsular substance comprised, at least in part, of a mucopolysaccharide or a mucopolysaccharide-protein complex.

Cytochemical localization of acid phosphatases in the dimorphic fungusSporothrix schenckii

A modified Gomori procedure at the electron microscopic level revealed a multiplicity of acid phosphatase activity sites in both yeast-like and mycelial phase cells. Vacuoles and the periplasmic space contained electron opaque deposits (lead phosphate) that were absent in control incubations either lacking the substrate (β-glycerophosphate) or fortified with an inhibitor (sodium fluoride). The outermost region of the cell envelope was also active and, in contradistinction to previous examples with other yeasts, deposition of lead phosphate in this locale occurred even when the rate of orthophosphate generation was drastically reduced by lowering the substrate concentration. When mechanically disrupted yeast-like cells were washed and then subjected to the cytochemical procedure, pieces of broken cell envelope gave a positive reaction. The reaction product was invariably restricted to one side of cell wall cross sections. A specific and novel association of acid phosphatase with a microfibrillar zone was indicated.

Scanning-beam electron microscopy of the conidia of the brown and albino filamentous varieties of Histoplasma capsulatum

Aspects of the surface appearance and external morphology of the conidial forms of the albino and brown filamentous varieties ofHistoplasma capsulatum as seen by scanning-beam electron microscopy are described and illustrated by electron micrographs. Septal areas between the hyphal cells of the supporting mycelium are seen as slightly elevated annular rings or ridges. The smooth micro- and macroconidia of the albino filamentous variety show a fine wrinkling and delicate irregularities of surface texture. Macroconidia of the brown filamentous variety are illustrated showing variations in numbers and respective length of the conspicuous wall projections or tubercles. The 3-dimensional perspective, unusual depth of focus, and high resolving power of the stereoscan technique permitted observations of external conidia morphology unattainable by other methods of study.

Drug—induced alterations in the ultrastructural organization of Histoplasma capsulatum and Blastomyces dermatitidis

Aspects of the fine structure as seen in thin section of yeastlike cells ofHistoplasma capsulatum andBlastomyces dermatitidis exposed to polyenic antibiotics are described and illustrated by electron micrographs. The exposure of log phase yeastlike cells to minimal fungicidal concentrations of both amphotericin B (Fungizone) and hamycin resulted in detectable alterations of the plasma membrane, and, to a lesser extent, the mitochondria. WithH. capsulatum, ultrastructural changes were observed to occur within 1 h exposure to amphotericin B. Marked degenerative changes and plasmolysis were observed to occur within 6 hrs exposure of the yeastlike cells to both polyenes. The observed changes in ultrastructural appearance are compatible with the concept of binding of the polyene with membrane sterol and subsequent damage due to alterations of permeability.