Specific digestive deficiency of phagocytes in paracoccidioidomycosis. Its absence in peripheral blood neutrophils of members of the nuclear family of patients. An initial report.

Paracoccidioidomycosis (PARA) affects only a minority of individuals, who have presumably been exposed to the causative fungus (Paracoccidioides brasiliensis). Neutrophils (PMNs) from patients with PARA show a significant and specific digestive deficiency phagocytosed P. brasiliensis in vitro. It is not known whether the defect is acquired after contact with the fungus, or precedes it. We studied the spouses and offspring of three patients with PARA. Individuals studied stayed in the same house as their husband or father. None of the relatives had evidence of PARA, and their PMNs showed no defect in their ability to digest or kill the fungus. Relatives showed no indication of sensitization against P. brasiliensis. These results are compatible with the view that effective contact with P. brasiliensis may occur only under restricted conditions and that the defect in digestive ability is an infrequent and specific occurrence.

Relationship between digestive and killing abilities of neutrophils against Paracoccidioides brasiliensis.

Peripheral blood neutrophils (PMN) from patients with paracoccidioidomycosis killed and digested Paracoccidioides brasiliensis much less than did PMN from normal individuals or from patients with other diseases. However, deficiency in killing ability was less specific than digestive deficiency and correlated poorly with it. We conclude that the capacities of PMN to digest and kill P. brasiliensis are not intimately related phenomena, and that in paracoccidioidomycosis the key deficiency of neutrophil function is that of digestion of P. brasiliensis.

Influence of serum on in vitro digestion of Paracoccidioides brasiliensis by neutrophils.

Serum from patients with paracoccidioidomycosis (PARA) did not block digestive abilities of neutrophils (PMNs) from healthy individuals against Paracoccidioides brasiliensis. Conversely, serum from healthy donors did not enhance digestive capacities of PMNs from patients with PARA vis á vis the causative organism. We conclude that the specific digestive defect present in PMNs from patients with PARA is not mediated by serum factors.

Functions of polymorphonuclear leukocytes and individuality of Jorge Lobo's disease: absence of the specific leukocyte digestive defect against Paracoccidioides brasiliensis.

Peripheral blood neutrophils (PMNs) from a patient with Jorge Lobo's disease (JLD) digested well phagocytosed Paracoccidioides brasiliensis. We found no circulating antibodies against P. brasiliensis in the patient's serum. Such neutrophils showed myeloperoxidase activity and also digested normally phagocytosed Candida albicans. We had previously reported the presence of a specific digestive deficiency of PMNs from patients with paracoccidioidomycosis (PARA) vis à vis P. brasiliensis. Current findings provide new information about leukocyte functions in JLD and bolster the view that JLD, PARA and their respective causative microorganisms are distinct.

Polymorphonuclear leukocyte functions in psoriasis.

Circulating polymorphonuclear leukocyte (CPMN) functions were studied in patients with widespread psoriasis as well as in persons with chronic alcoholic liver disease (CALD), paracoccidioidomycosis, diverse granulomatous diseases, and normal individuals. We were unable to find stimulation or increase in CPMN functions in patients with psoriasis compared to normal individuals. Leukocytes from individuals with CALD had a lowering of their metabolic activation, chemotaxis, random movement, and adherence. CPMNs from patients with paracoccidioidomycosis showed a significant deficiency in their ability to digest Paracoccidioides brasiliensis. Our results are against the concept that functions of circulating PMNs are stimulated in psoriatics.

A novel method for estimating killing ability and digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

We describe a novel method by which phagocytosis, digestion and killing of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated simultaneously. Suspensions of P. brasiliensis (yeast-like phase) were sonicated, counted and incubated at 37 degrees C with known numbers of phagocytes. Control preparations contained no phagocytic cells. At given intervals samples were incorporated into gelatin nutrient medium and droplets of the mixtures were incubated at room temperature. Live yeast-like P. brasiliensis germinate in vitro and produce filaments. After incubation, droplets may be melted and examined under phase contrast optics, or the cells may be washed and stained by a variation of Papanicolaou's method. Digested P. brasiliensis, intact but non-germinating yeasts and filamented (viable) yeasts may be identified and counted. Killing and digestive abilities of phagocytes may be estimated by the difference between values obtained from phagocyte-containing and control preparations.

Functions of neutrophils in endemic Chediak-Higashi syndrome.

Endemic Chediak-Higashi Syndrome occurs in a restricted geographic area (Pregonero, State of Táchira, Venezuela). Neutrophils from these patients were unable to digest Candida albicans in vitro, but showed normal or increased metabolic activities. This finding supports the view that the endemic syndrome is bona fide Chediak-Higashi Syndrome.

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimated viability by means of vital dyes. Methylene blue Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans. Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium. In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25%. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.

Defect of in vitro digestive ability of polymorphonuclear leukocytes in paracoccidioidomycosis.

Selected functions of polymorphonuclear leukocytes were studied in patients with paracoccidioidomycosis (South American blastomycosis), in healthy control individuals, and in patients with diseases unrelated to paracoccidioidomycosis. Patients with paracoccidioidomycosis were also evaluated by standard immunological techniques. Phagocytosis and digestion of Paracoccidioides brasiliensis yeastlike cells in vitro was estimated by an original method. It was based on the appearance of phagocytosed P. brasiliensis in preparations stained by a modification of the Papanicolaou method and examined with phase-contrast optics. Interpretation of such findings was confirmed by electron microscopy. Two strains of P. brasiliensis were used. Strain 8506 was freshly isolated from a patient. Strain Pb9 was known to be nonpathogenic and to have a peculiar cell wall composition. Yeastlike cells of the Pb9 strain were digested significantly better than those of strain 8506. A higher number of leukocytes per fungus cells led to a higher proportion of digested P. brasiliensis. Leukocytes from patients with paracoccidioidomycosis phagocytosed the fungus in a normal way, but had a significant lower ability to digest it in vitro. When individual cases were analyzed, there was an excellent correlation between clinical evolution and digestive ability of polymorphonuclear leukocytes. There was good correlation between both of these and immunological parameters. Leukocytes from all groups behaved comparably in tests of general leukocyte function and in their abilities to kill and digest Candida albicans. Our results indicate that, as a group, polymorphonuclear leukocytes from patients with paracoccidioidomycosis had a significant, rather specific, defect in their in vitro digestive capacity against phagocytosed P. brasiliensis. There was also an inverse correlation between strain pathogenicity and its susceptibility to in vitro digestion by polymorphonuclear leukocytes. Our findings are concordant with and relevant to clinical reality.

New method for estimating digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

We describe a method by which phagocytosis and digestion of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated. Suspensions of P. brasiliensis in its yeastlike phase were sonicated, counted, and incubated with known numbers of peripheral blood polymorphonuclear leukocytes. At given intervals, cytocentrifuge droplets were stained by a variation of Papanicolaou's method. Stained preparations were examined with phase-contrast optics. Digested organisms showed total or partial disappearance of protoplasm. Green-stained cell walls resisted digestion. The proportion of digested cells as a function of time was estimated.