The diagnosis of Histoplasma capsulatum infection by serologic testing for the presence of antibodies is limited by a high rate of false positive and false negative results and by the requirement that the patient have a normal immune response. We have developed a radioimmunoassay for the detection of H. capsulatum antigen in urine and serum specimens. Antigenuria was noted in 20 of 22 episodes of disseminated histoplasmosis that occurred in 16 patients, in 6 of 32 patients with self-limited infection, in 2 of 32 patients with cavitary histoplasmosis, and in 4 of 8 patients with a sarcoid-like illness caused by H. capsulatum. The detection of antigen in urine was reproducible in 38 of 41 (93 percent) retests of specimens. H. capsulatum antigen was also detected in the serum during 11 of the 22 episodes of disseminated histoplasmosis, in none of the 12 episodes of other types of histoplasmosis in patients with antigenuria, in 1 of the 33 patients with histoplasmosis who lacked the urinary antigen, and in none of the 50 controls. Antigenemia and antigenuria decreased after initiation of antifungal therapy and recurred in patients who had a relapse. We conclude that this radioimmunoassay for H. capsulatum antigen represents a useful new method for the rapid diagnosis of disseminated histoplasmosis.

While laccase of Cryptococcus neoformans is implicated in the virulence of the organism, our recent studies showing absence of melanin in the infected mouse brain has led us to a search for alternative roles for laccase in cryptococcosis. We investigated the role of laccase in protection of C. neoformans against murine alveolar macrophage (AM)-mediated antifungal activity by using a pair of congenic laccase-positive (2E-TUC) and laccase-deficient (2E-TU) strains. The laccase-positive cells with laccase derepression were more resistant to the antifungal activity of AM than a laccase-deficient strain ([28.9 ± 1.2]% versus [40.2 ± 2.6]% killing). Addition of l-dopa toCryptococcus to produce melanin in a laccase-positive strain resulted in a slight increase in protection of C. neoformans from the antifungal activity of macrophages ([25.4 ± 3.4]% versus [28.9 ± 1.2]% killing). Recombinant cryptococcal laccase exhibited iron oxidase activity in converting Fe(II) to Fe(III). Moreover, recombinant laccase inhibited killing of C. neoformans by hydroxyl radicals catalyzed by iron in a cell-free system. Addition of the hydroxyl radical scavenger mannitol or dimethyl sulfoxide to AMs prior to the introduction of cryptococcal cells decreased killing of both strains and reduced the difference in susceptibility between the laccase-positive and laccase-deficient strains. Furthermore, laccase-mediated protection from AM killing was inhibited by the addition of Fe(II), presumably by overcoming the effects of the iron oxidase activity of cryptococcal laccase. These results suggest that the iron oxidase activity of laccase may protect C. neoformans from macrophages by oxidation of phagosomal iron to Fe(III) with a resultant decrease in hydroxyl radical formation.

The lymphoid cells responsible for protective immunity to histoplasmosis were characterized. Adoptive transfer of spleen and peritoneal cells treated with antiserum to theta-antigen from mice immunized with ribosomes or live yeast cells of Histoplasma capsulatum abrogated the ability of these cells to protect the syngeneic recipients, whereas treatment of lymphoid cells with antiserum to IgG did not affect the immunity. Prior removal of glass-adhering cells from spleen and peritoneal cell suspensions did not alter their protective activity. Treatment with mitomycin C, an antimitotic agent, ablated the capacity of immune lymphocytes to protect the syngeneic recipients. These results indicate that the immune spleen and peritoneal cells that confer immunity to histoplasmosis are thymus-dependent (T) lymphocytes and that their active proliferation in the recipients is necessary for expression of the protective immunity. Furthermore, the immunity elicited by immunization with histoplasma ribosomes and live yeast cells is mediated by a similar mechanism.

Immunization with ribosomal preparations from Haemophilus influenzae type b elicited protective immunity in mice. Ribosomes from disrupted cells where isolated by differential centrifugation using sodium dodecyl sulfate. The washed ribosomes contained 25% protein and 75% ribonucleic acid and sedimented as a single peak on sucrose density gradient analysis with a sedimentation coefficient of 67S, using Escherichia coli ribosomes as a 70S marker. Immunodiffusion tests with antipolyribose phosphate serum showed that the ribosomes were free from capsular material. Mice immunized subcutaneously with ribosomes, with or without adjuvant, were challenged intraperitoneally with 100 to 1,000 mean lethal doses of H. influenzae type b suspended in gastric mucin. Significant protection was induced by ribosomes and was compared to that obtained after sublethal infection with live cells. The protection was greatly enhanced after incorporation of ribosomes into adjuvants. Maximum protection (90 to 95%) was observed at 1 to 2 weeks after immunization. Ribosomes from a nonencapsulated strain of H. influenzae were as immunogenic as those from the encapsulated strain, demonstrating that the capsular material is not responsible for immunogenicity of Haemophilus ribosomes.

No abstract