Clinico-pathological studies on experimental cryptococcal mastitis in goats

Unilateral intramammary inoculation of 10 goats withCryptococcus neoformans (2×10⁶ yeast cells) resulted in the development of mastitis, with gross and microscopic lesions being restricted to the infected udder halves only and there was no dissemination of infection to the opposite uninfected udder halves as well as to other organs of the body. The experiment was continued for 40 days, with 2 animals each from the infected and control groups being killed on 5th, 10th, 20th, 30th and 40th day postinoculation (DPI). Initial enlargement of the infected udder halves was followed by marked decrease in size leading to very small, firm and nodular udder halves. After infection, there was also sharp fall in the milk yield. Cryptococcal organisms were demonstrated in the mastitic milk and udder impression smears with special stains.C. neoformans was reisolated from the milk of only infected udder halves up to 25th DPI. Microsopically, there was initially acute diffuse purulent mastitis which later on became chronic, characterised by marked infiltration of lymphocytes, macrophages, extensive fibrosis and development of multiple granulomas. The cryptococcal organisms could be demonstrated in the udder sections only up to 30th DPI. It is concluded that intramammary inoculation ofCryptococcus neoformans in goats leads to severe mastitis with sharp fall in milk yield.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

High detection rates of cryptococcal antigen in pulmonary cryptococcosis by Eiken Latex Agglutination test with pronase pretreatment

Two different kits for the detection of serum cryptococcal antigen in patients with pulmonary cryptococcosis were evaluated. The Eiken test (the Eiken Co., Tokyo), which uses pronase for pretreatment of serum, was compared with the Crypto-LA test (International Biological Laboratories, Cranbury, NJ), which did not use pronase prior to testing. Cryptococcal antigen was detected in 21 of 23 patients (91%) with the Eiken test and in only 10 of 23 patients (43%) with the Crypto-LA test (p<0.01 by Mcnemar test). However, the sensitivity of two tests was identical without use of pronase, as both tests could detect as little as 10⁴ cells/ml ofCryptococcus neoformans and 10 ng/ml of capsular polysaccharide ofC. neoformans. In those serum specimens for which both tests were positive, titers were much higher for the Eiken test, but there was a statistically significant correlation between the two tests (coefficient correlation 0.79,p<0.01). Cryptococcal antigen titer levels measured by the Eiken test correlated well with clinical courses. There was one false-positive reaction among 82 sera of non-cryptococcal patients. Pronase enhanced the sensitivity of the Eiken test, which appeared to be useful in patients with pulmonary cryptococcal disease, and its use may prevent unneeded lung biopsies.     

Intravascular granuloma induced by intravenous inoculation ofCryptococcus neoformans

In rodents an intravenous administration of viableCryptococcus (C.) neoformans cells frequently resulted in attachment of intravascular cryptococcal granulomas to inner walls of the large to medium-sized veins of various organs, including the lungs, liver and spleen. In order to elucidate the pathogenesis of granulomatous changes, the cells composing the intravascular granulomas were observed by electron microscopic peroxidase (PO) cytochemistry. The granuloma composing cells could be divided into the following four types according to the pattern of endogenous peroxidase activity: exudate macrophage (Mφ, type I), PO-negative Mφ (type II), resident Mφ (type III) and other inflammatory cells (type IV). In the intravenous granulomas of the lung, the percentages of composed cells were 39.0% for type I, 57.9% for type II, 0% for type III and 3.1% for type IV. By contrast, in the interstitial granulomas in the lung, type III Mφs, possibly derived from alveolar Mφs, played a significant role in granuloma formation. This may indicate that the intravascular granuloma is almost composed of macrophages derived from monocytes rather than alveolar macrophages. The expression of ICAM-1 on endothelia of the pulmonary veins was examined by immunoelectron microscopy. An immunogold labeling index was significantly augmented on the surface of endothelia in response to intravenous challenge ofC. neoformans. The intravascular granuloma demonstrates that the monocytes develop into the granuloma-composing macrophages and suppress the cryptococcal activities even hi the peripheral blood resulting in an assistance of endothelial functions.     

In vivo and in vitro studies with an atypical,rhinotropic isolate of Cryptococcus neoformans

An atypical isolate of Cryptococcus neoformans was investigated because of its consistent and reproducible production of gross nasal pathology following i.v. injection in Swiss albino mice. Dose response to graded concentrations ranging from 1×l0²–1×l0⁷ cells/mouse yielded an LD₅₀ of 1.4×10³ cells/mouse for the atypical rhinotropic strain H140 which was significantly less virulent (p<0.01) than our reference strain of Cryptococcus neoformans. There was no significant difference in mortality following the injection of in vitro vs. in vivo passed inoculum. As early as two weeks after inoculation, this strain produced gross nasal enlargement to approximately 2–3 × normal dimensions with granulomatous and ulcerated lesions. The LD₆₀ resulted in the greatest percentage of nasal involvement (85%). C. neoformans was demonstrated by culture and histopathology in the noses, brains, lungs, livers and kidneys. A temperature selection was indicated by findings of a lower temperature minimum for subcultures isolated from the noses relative to those isolated from the brain, and by the fact that the most densely populated organs following intraperitoneal injection were the testes. This route of inoculation resulted in cutaneous nasal involvement in a manner analogous to that following i.v. injection. The atypical isolate was unable to assimilate trehalose or raffinose but otherwise was entirely consistent with identification as C. neoformans and produced characteristic CNS and general organ system disease in addition to the rhinotropic cutaneous manifestations. The model characterized here in normal mice may be of value in studies of fungal dermotropism.     

Olfactory mucosa response in guinea pigs following intranasal instillation withCryptococcus neoformans

A central nervous system isolate from an acquired immunodeficiency syndrome (AIDS) patient of 10³ Cryptococcus neoformans cells was instilled intranasally into guinea pigs. These were killed to evaluate the fate of the organisms and the response of the olfactory mucosa. Olfactory epithelium prevented the penetration ofCryptococcus neoformans and showed focal hyperplastic responses. The organisms, which manifested an affinity for the olfactory portion of the nasal cavities, were cleared from the olfactory space with no intervention from the immune system cells. By the end of the fifth week almost no organisms could be found and there was no histological evidence of dissemination. In contrast, destruction of the olfactory epithelium with zinc sulfate allowed the invasion of the subepithelial tissues, demonstrating the role of the olfactory mucosa in preventing infection withCryptococcus neoformans through the nasal route. The results and the model described in this report may be useful in clarifying the pathogenic mechanisms of cryptococcosis and the non immune mediated host responses toCryptococcus neoformans.     

Muscular-skeletal cryptococcosis in a patient with idiopathic CD4+ lymphopenia

A healthy 27-year-old woman presented, four months after childbirth, ingravescent pain and claudication of the left lower limb. Magnetic Resonance Imaging of the lumbosacral and iliac regions showed widespread muscular-skeletal lesions. The patient underwent surgery; Cryptococcus neoformans was isolated from surgical samples. Liposomal amphotericin B, fluconazole and itraconazole were administered. Laboratory findings showed lymphocytopenia, with reduction of CD4+ lymphocytes (23 cells per cubic millimeter) in the absence of HIV infection and any other defined immunodeficiency. This is a rare case of muscular-skeletal cryptococcal infection isolated in a subject affected with idiopathic CD4+ lymphocytopenia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue changes in cryptococcosis: histologie alteration from gelatinous to suppurative granulomatous tissue response with asteroid body

The histologic variety and transformation in cutaneous cryptococcosis with acute lymphocytic leukemia before antifungal treatment and after the start of treatment were studied by the light and electron microscopic examinations. The initial cutaneous lesions before treatment revealed gelatinous tissue reactions, and Cryptococcus neoformans (serotype A) were isolated from the skin biopsy specimen and blood. However, later recurrent cutaneous lesions receiving antifungal treatment revealed suppurative granulomatous tissue reactions, and fungal cultures of the skin biopsy specimen changed to negative even though numerous yeasts stained with PAS were observed in skin lesions. Moreover, in the later lesion a few giant cells contained asteroid bodies without central spores. Ultrastructure of the later cutaneous lesions is presented.     

On the phylogenetic relationship between Lipomyces and Cryptococcus

Summary Additional evidence is presented which confirms previous suggestions that there may not be a phylogenetic relationship between the imperfect genusCryptococcus and the perfect genusLipomyces. This evidence is based on several observations: (1) that oxomalonic (mesoxalic) acid can be identified in media in whichLipomyces lipofer andL. starkeyi are growing but not in media in whichCryptococcus neoformans is growing (2) that dividing nuclei in cells ofC. neoformans do not appear to behave in a manner similar to those described in cells ofL. lipofer and (3) that attempts to obtain. ascospore formation inC. neoformans have so far been unsuccessful.The latter stages of these investigations were supported by a Public Health Research Grant (Project 605-7-240) to the author.     

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

Ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid of a patient with cryptococcal meningitis

The ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid, which was obtained from a patient suffering from systemic lupus erythematosus and cryptococcal meningitis, before treatment and on the 10th and 20th day after the start of treatment was studied. Numerous cryptococci were detected in the cerebrospinal fluid before treatment. However, in most of them their biological activity was low and their organelles were not clear. A few yeasts preserved well their organelles. Such cells showed a tendency to develop vesicular membrane structures (lomasomes) touching the internal part of the cell wall. In the cerebrospinal fluid on the 20th day all of the yeasts were dead, even though numerous yeasts were observed by an India-ink method. At this time no colonies were recovered from the fluid.     

Cryptococcus neoformàns var. gattii among patients with cryptococcal meningitis in Mexico. First observations

A retrospective study of 20 patients with cryptococcal meningitis and their isolated strains was performed. Cryptococcus neoformans var. neoformans was recovered from 14 (70%) cases, and var. gattii was recovered from six (30%) patients. Twelve patients had AIDS (all carrying var. neoformans), two had other diseases (one with var. neoformans and one var. gattii) and there was no identifiable underlying disease in six (one var. neoformans and five var. gattii). Fourteen patients (11 var. neoformans and three var. gattii) resided in the Mexico City area, where a temperate climate is prevalent, and there were six cases (three var. neoformans and three var. gattii) from states with a tropical/subtropical climate. Although there was no significant statistical difference between the two varieties, the fatal outcome was higher in patients with var. neoformans. The disease caused by var. gattii strains was characterized by a higher opening pressure, more inflamatory changes of CSF and a longer clinical course (delayed clinical and mycological cure). Cryptococcus neoformans var. gattii is a significant cause of cryptococcal meningitis in patients without underlying diseases in Mexico.     

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD₅₀ value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.     

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis

Toll‐like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT‐PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti‐TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre‐incubation of PBMCs from healthy donors with anti‐TLR2 led to reduced expression of IFN‐γ and IL‐12p70, but not of IL‐4 and IL‐10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.     

Effect of amphotericin B on the lipids of five different strains ofCryptococcus neoformans

Cells of five strains ofCryptococcus neoformans were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectra. Such determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimal inhibitory concentration) levels. Marked alterations of the lipid and sterol contents were observed in the amphotericin B — treated cells. Moreover, ergosterol disappeared in these antibiotic-exposed cells. It is concluded that amphotericin B altered the lipid profiles, especially sterols ofC. neoformans.     

Cryptococcus neoformans-Derived Microvesicles Enhance the Pathogenesis of Fungal Brain Infection

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans in vitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.     

Species discrimination and concept formation by rhesus monkeys (Macaca mulatta)

There are 19 species in genusMacaca and some of them are living in sympatry (Fooden, 1980). Although inter-specific hybrids are relatively easy to produce under artificial conditions, hybridization does not occur naturally. What is preventing that among the species of genusMacaca? Three rhesus monkeys acquired a discrimination between pictures with rhesus monkeys and without rhesus monkeys. All subjects showed positive transfer of this discrimination to new pictures with rhesus monkeys and without rhesus monkeys. A further test showed that these monkeys could discriminate between pictures of rhesus monkeys and pictures of Japanese monkeys. The results suggest that rhesus monkeys recognize rhesus monkeys as a class, independent of the actual stimuli such as a picture or an individual monkey. The ability to recognize members of their own species and the opportunities for such learning may be an important factor preventing hybridization among the species of genusMacaca.